Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

International Nuclear Information System (INIS)

Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

2014-01-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM TGF , FCM PDGF ) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM B ). FCM TGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM TGF ≫FCM PDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM TGF >FCM PDGF ) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify

3,3'Diindolylmethane suppresses vascular smooth muscle cell phenotypic modulation and inhibits neointima formation after carotid injury.

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Hongjing Guan

Full Text Available 3,3'Diindolylmethane (DIM, a natural phytochemical, has shown inhibitory effects on the growth and migration of a variety of cancer cells; however, whether DIM has similar effects on vascular smooth muscle cells (VSMCs remains unknown. The purpose of this study was to assess the effects of DIM on the proliferation and migration of cultured VSMCs and neointima formation in a carotid injury model, as well as the related cell signaling mechanisms.DIM dose-dependently inhibited the platelet-derived growth factor (PDGF-BB-induced proliferation of VSMCs without cell cytotoxicity. This inhibition was caused by a G0/G1 phase cell cycle arrest demonstrated by fluorescence-activated cell-sorting analysis. We also showed that DIM-induced growth inhibition was associated with the inhibition of the expression of cyclin D1 and cyclin-dependent kinase (CDK 4/6 as well as an increase in p27(Kip1 levels in PDGF-stimulated VSMCs. Moreover, DIM was also found to modulate migration of VSMCs and smooth muscle-specific contractile marker expression. Mechanistically, DIM negatively modulated PDGF-BB-induced phosphorylation of PDGF-recptorβ (PDGF-Rβ and the activities of downstream signaling molecules including Akt/glycogen synthase kinase(GSK3β, extracellular signal-regulated kinase1/2 (ERK1/2, and signal transducers and activators of transcription 3 (STAT3. Our in vivo studies using a mouse carotid arterial injury model revealed that treatment with 150 mg/kg DIM resulted in significant reduction of the neointima/media ratio and proliferating cell nuclear antigen (PCNA-positive cells, without affecting apoptosis of vascular cells and reendothelialization. Infiltration of inflammatory cells was also inhibited by DIM administration.These results demonstrate that DIM can suppress the phenotypic modulation of VSMCs and neointima hyperplasia after vascular injury. These beneficial effects on VSMCs were at least partly mediated by the inhibition of PDGF-Rβ and the

Modulation of phenotypic and functional maturation of murine dendritic cells (DCs) by purified Achyranthes bidentata polysaccharide (ABP).

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Zou, Yaxuan; Meng, Jingjuan; Chen, Wenna; Liu, Jingling; Li, Xuan; Li, Weiwei; Lu, Changlong; Shan, Fengping

2011-08-01

There are a large number of interactions at molecular and cellular levels between the plant polysaccharides and immune system. Plant polysaccharides present an interesting effects as immunomodulators, particularly in the induction of the cells both in innate and adaptive immune systems. Activation of DCs could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. ABP is a purified polysaccharide isolated from Achyranthes bidentata, a traditional Chinese medicine (TCM). The aim of this study is to investigate modulation of phenotypic and functional maturation of murine DCs by ABP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DC, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acidic phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. In fact, we found that purified ABP induced phenotypic maturation revealed by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside DCs (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). Finally, ABP increased the production of IL-12. These data reveal that ABP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting. It is therefore concluded that ABP can exert positive modulation to murine DCs. Copyright © 2011 Elsevier B.V. All rights reserved.

Antithymocyte Globulin Induces a Tolerogenic Phenotype in Human Dendritic Cells

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Tobias Roider

2016-12-01

Full Text Available Antithymocyte globulin (ATG is used in the prevention of graft-versus-host disease during allogeneic hematopoietic stem cell transplantation. It is generally accepted that ATG mediates its immunosuppressive effect primarily via depletion of T cells. Here, we analyzed the impact of ATG-Fresenius (now Grafalon® on human monocyte-derived dendritic cells (DC. ATG induced a semi-mature phenotype in DC with significantly reduced expression of CD14, increased expression of HLA-DR, and intermediate expression of CD54, CD80, CD83, and CD86. ATG-DC showed an increase in IL-10 secretion but no IL-12 production. In line with this tolerogenic phenotype, ATG caused a significant induction of indoleamine 2,3-dioxygenase expression and a concomitant increase in levels of tryptophan metabolites in the supernatants of DC. Further, ATG-DC did not induce the proliferation of allogeneic T cells in a mixed lymphocyte reaction but actively suppressed the T cell proliferation induced by mature DC. These data suggest that besides its well-known effect on T cells, ATG modulates the phenotype of DC in a tolerogenic way, which might constitute an essential part of its immunosuppressive action in vivo.

Molecular Mechanisms Modulating the Phenotype of Macrophages and Microglia

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Stephanie A. Amici

2017-11-01

Full Text Available Macrophages and microglia play crucial roles during central nervous system development, homeostasis and acute events such as infection or injury. The diverse functions of tissue macrophages and microglia are mirrored by equally diverse phenotypes. A model of inflammatory/M1 versus a resolution phase/M2 macrophages has been widely used. However, the complexity of macrophage function can only be achieved by the existence of varied, plastic and tridimensional macrophage phenotypes. Understanding how tissue macrophages integrate environmental signals via molecular programs to define pathogen/injury inflammatory responses provides an opportunity to better understand the multilayered nature of macrophages, as well as target and modulate cellular programs to control excessive inflammation. This is particularly important in MS and other neuroinflammatory diseases, where chronic inflammatory macrophage and microglial responses may contribute to pathology. Here, we perform a comprehensive review of our current understanding of how molecular pathways modulate tissue macrophage phenotype, covering both classic pathways and the emerging role of microRNAs, receptor-tyrosine kinases and metabolism in macrophage phenotype. In addition, we discuss pathway parallels in microglia, novel markers helpful in the identification of peripheral macrophages versus microglia and markers linked to their phenotype.

Inflammatory cell phenotypes in AAAs: their role and potential as targets for therapy.

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Dale, Matthew A; Ruhlman, Melissa K; Baxter, B Timothy

2015-08-01

Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused ≈15 000 deaths annually in the United States. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4(+) T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Proinflammatory CD4(+) T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to proinflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the proinflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention. © 2015 American Heart Association, Inc.

Inflammatory cell phenotypes in AAAs; their role and potential as targets for therapy

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Dale, Matthew A; Ruhlman, Melissa K.; Baxter, B. Timothy

2015-01-01

Abdominal aortic aneurysms are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused approximately 15,000 deaths annually in the U.S. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4+ T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Pro-inflammatory CD4+ T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to pro-inflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the pro-inflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention. PMID:26044582

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

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Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

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Wang, Huang-Joe; Chen, Sung-Fang; Lo, Wan-Yu

2015-05-01

The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software. A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

The phenotypic plasticity of developmental modules

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Aabha I. Sharma

2016-08-01

Full Text Available Abstract Background Organisms develop and evolve in a modular fashion, but how individual modules interact with the environment remains poorly understood. Phenotypically plastic traits are often under selection, and studies are needed to address how traits respond to the environment in a modular fashion. In this study, tissue-specific plasticity of melanic spots was examined in the large milkweed bug, Oncopeltus fasciatus. Results Although the size of the abdominal melanic bands varied according to rearing temperatures, wing melanic bands were more robust. To explore the regulation of abdominal pigmentation plasticity, candidate genes involved in abdominal melanic spot patterning and biosynthesis of melanin were analyzed. While the knockdown of dopa decarboxylase (Ddc led to lighter pigmentation in both the wings and the abdomen, the shape of the melanic elements remained unaffected. Although the knockdown of Abdominal-B (Abd-B partially phenocopied the low-temperature phenotype, the abdominal bands were still sensitive to temperature shifts. These observations suggest that regulators downstream of Abd-B but upstream of DDC are responsible for the temperature response of the abdomen. Ablation of wings led to the regeneration of a smaller wing with reduced melanic bands that were shifted proximally. In addition, the knockdown of the Wnt signaling nuclear effector genes, armadillo 1 and armadillo 2, altered both the melanic bands and the wing shape. Thus, the pleiotropic effects of Wnt signaling may constrain the amount of plasticity in wing melanic bands. Conclusions We propose that when traits are regulated by distinct pre-patterning mechanisms, they can respond to the environment in a modular fashion, whereas when the environment impacts developmental regulators that are shared between different modules, phenotypic plasticity can manifest as a developmentally integrated system.

RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton.

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Tang, Lian; Dai, Fan; Liu, Yan; Yu, Xiaoqiang; Huang, Chao; Wang, Yuqin; Yao, Wenjuan

2018-05-20

The RhoA/ROCK signaling pathway regulates cell morphology, adhesion, proliferation, and migration. In this study, we investigated the regulatory role of RhoA/ROCK signaling on PDGF-BB-mediated smooth muscle phenotypic modulation and vascular remodeling and clarified the molecular mechanisms behind these effects. PDGF-BB treatment induced the activation of RhoA, ROCK, PDGF-Rβ, and the expression of PDGF-Rβ in HA-VSMCs (human aortic vascular smooth muscle cells). PDGF-Rβ inhibition and RhoA suppression blocked PDGF-BB-induced RhoA activation and ROCK induction. In addition, PDGF-BB-mediated cell proliferation and migration were suppressed by PDGF-Rβ inhibition, RhoA suppression, and ROCK inhibition, suggesting that PDGF-BB promotes phenotypic modulation of HA-VSMCs by activating the RhoA/ROCK pathway via the PDGF receptor. Moreover, suppressing both ROCK1 and ROCK2 blocked cell cycle progression from G0/G1 to S phase by decreasing the transcription and protein expression of cyclin D1, CDK2, and CDK4 via JNK/c-Jun pathway, thus reducing cell proliferation in PDGF-BB-treated HA-VSMCs. ROCK1 deletion, rather than ROCK2 suppression, significantly inhibited PDGF-BB-induced migration by reducing the expression of vimentin and preventing the remodeling of vimentin and phospho-vimentin. Furthermore, ROCK1 deletion suppressed vimentin by inhibiting the phosphorylation of Smad2/3 and the nuclear translocation of Smad4. These findings suggested that ROCK1 and ROCK2 might play different roles in PDGF-BB-mediated cell proliferation and migration in HA-VSMCs. In addition, PDGF-BB and its receptor participated in neointima formation and vascular remodeling by promoting cell cycle protein expression via the JNK pathway and enhancing vimentin expression in a rat balloon injury model; effects that were inhibited by treatment with fasudil. Together, the results of this study reveal a novel mechanism through which RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and

Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry.

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Giovanna Clavarino

Full Text Available A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45 and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.

Mesothelioma tumor cells modulate dendritic cell lipid content, phenotype and function.

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Joanne K Gardner

Full Text Available Dendritic cells (DCs play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay, upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+ CD8α- DCs, CD4- CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

Phenotype modulation of airway smooth muscle in asthma

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Wright, David B.; Trian, Thomas; Siddiqui, Sana; Pascoe, Chris D.; Johnson, Jill R.; Dekkers, Bart G. J.; Dakshinamurti, Shyamala; Bagchi, Rushita; Burgess, Janette K.; Kanabar, Varsha; Ojo, Oluwaseun O.

The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory

Stem Cell Microencapsulation for Phenotypic Control, Bioprocessing, and Transplantation

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Wilson, Jenna L.

2014-01-01

Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self-renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi-permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. PMID:23239279

Liver X Receptor Genes Variants Modulate ALS Phenotype.

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Mouzat, Kevin; Molinari, Nicolas; Kantar, Jovana; Polge, Anne; Corcia, Philippe; Couratier, Philippe; Clavelou, Pierre; Juntas-Morales, Raul; Pageot, Nicolas; Lobaccaro, Jean -Marc A; Raoul, Cedric; Lumbroso, Serge; Camu, William

2018-03-01

Amyotrophic lateral sclerosis (ALS) is one of the most severe motor neuron (MN) disorders in adults. Phenotype of ALS patients is highly variable and may be influenced by modulators of energy metabolism. Recent works have implicated the liver X receptors α and β (LXRs), either in the propagation process of ALS or in the maintenance of MN survival. LXRs are nuclear receptors activated by oxysterols, modulating cholesterol levels, a suspected modulator of ALS severity. In a cohort of 438 ALS patients and 330 healthy controls, the influence of LXR genes on ALS risk and phenotype was studied using single nucleotide polymorphisms (SNPs). The two LXRα SNPs rs2279238 and rs7120118 were shown to be associated with age at onset in ALS patients. Consistently, homozygotes were twice more correlated than were heterozygotes to delayed onset. The onset was thus delayed by 3.9 years for rs2279238 C/T carriers and 7.8 years for T/T carriers. Similar results were obtained for rs7120118 (+2.1 years and +6.7 years for T/C and C/C genotypes, respectively). The LXRβ SNP rs2695121 was also shown to be associated with a 30% increase of ALS duration (p = 0.0055, FDR = 0.044). The tested genotypes were not associated with ALS risk. These findings add further evidence to the suspected implication of LXR genes in the disease process of ALS and might open new perspectives in ALS therapeutics.

Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

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Kun Taek Park

Full Text Available Phylogenic comparisons of the mononuclear phagocyte system (MPS of humans and mice demonstrate phenotypic divergence of dendritic cell (DC subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2, plasmacytoid DC (pDC, and monocyte derived DC (MoDC. DC of Artiodactyla (pigs and ruminants can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC. Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ. Functional analysis revealed each of these populations can take up and process antigens (Ags, present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

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2017-09-01

AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...CA130273 - Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM...hypothesis, we originally proposed to transform neural stem cells (NSCs) and neural progenitor cells (NPCs) in vivo by expressing an activated form

A mathematical model of cancer cells with phenotypic plasticity

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Da Zhou

2015-12-01

Full Text Available Purpose: The phenotypic plasticity of cancer cells is recently becoming a cutting-edge research area in cancer, which challenges the cellular hierarchy proposed by the conventional cancer stem cell theory. In this study, we establish a mathematical model for describing the phenotypic plasticity of cancer cells, based on which we try to find some salient features that can characterize the dynamic behavior of the phenotypic plasticity especially in comparison to the hierarchical model of cancer cells. Methods: We model cancer as population dynamics composed of different phenotypes of cancer cells. In this model, not only can cancer cells divide (symmetrically and asymmetrically and die, but they can also convert into other cellular phenotypes. According to the Law of Mass Action, the cellular processes can be captured by a system of ordinary differential equations (ODEs. On one hand, we can analyze the long-term stability of the model by applying qualitative method of ODEs. On the other hand, we are also concerned about the short-term behavior of the model by studying its transient dynamics. Meanwhile, we validate our model to the cell-state dynamics in published experimental data.Results: Our results show that the phenotypic plasticity plays important roles in both stabilizing the distribution of different phenotypic mixture and maintaining the cancer stem cells proportion. In particular, the phenotypic plasticity model shows decided advantages over the hierarchical model in predicting the phenotypic equilibrium and cancer stem cells’ overshoot reported in previous biological experiments in cancer cell lines.Conclusion: Since the validity of the phenotypic plasticity paradigm and the conventional cancer stem cell theory is still debated in experimental biology, it is worthy of theoretically searching for good indicators to distinguish the two models through quantitative methods. According to our study, the phenotypic equilibrium and overshoot

Galunisertib suppresses the staminal phenotype in hepatocellular carcinoma by modulating CD44 expression.

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Rani, Bhavna; Malfettone, Andrea; Dituri, Francesco; Soukupova, Jitka; Lupo, Luigi; Mancarella, Serena; Fabregat, Isabel; Giannelli, Gianluigi

2018-03-07

Cancer stem cells (CSCs) niche in the tumor microenvironment is responsible for cancer recurrence and therapy failure. To better understand its molecular and biological involvement in hepatocellular carcinoma (HCC) progression, one can design more effective therapies and tailored then to individual patients. While sorafenib is currently the only approved drug for first-line treatment of advanced stage HCC, its role in modulating the CSC niche is estimated to be small. By contrast, transforming growth factor (TGF)-β pathway seems to influence the CSC and thus may impact hallmarks of HCC, such as liver fibrosis, cirrhosis, and tumor progression. Therefore, blocking this pathway may offer an appealing and druggable target. In our study, we have used galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-β receptor I (TGFβI/ALK5) activation, currently under clinical investigation in HCC patients. Because the drug resistance is mainly mediated by CSCs, we tested the effects of galunisertib on stemness phenotype in HCC cells to determine whether TGF-β signaling modulates CSC niche and drug resistance. Galunisertib modulated the expression of stemness-related genes only in the invasive (HLE and HLF) HCC cells inducing a decreased expression of CD44 and THY1. Furthermore, galunisertib also reduced the stemness-related functions of invasive HCC cells decreasing the formation of colonies, liver spheroids and invasive growth ability. Interestingly, CD44 loss of function mimicked the galunisertib effects on HCC stemness-related functions. Galunisertib treatment also reduced the expression of stemness-related genes in ex vivo human HCC specimens. Our observations are the first evidence that galunisertib effectiveness overcomes stemness-derived aggressiveness via decreased expression CD44 and THY1.

Posterior association networks and functional modules inferred from rich phenotypes of gene perturbations.

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Xin Wang

Full Text Available Combinatorial gene perturbations provide rich information for a systematic exploration of genetic interactions. Despite successful applications to bacteria and yeast, the scalability of this approach remains a major challenge for higher organisms such as humans. Here, we report a novel experimental and computational framework to efficiently address this challenge by limiting the 'search space' for important genetic interactions. We propose to integrate rich phenotypes of multiple single gene perturbations to robustly predict functional modules, which can subsequently be subjected to further experimental investigations such as combinatorial gene silencing. We present posterior association networks (PANs to predict functional interactions between genes estimated using a Bayesian mixture modelling approach. The major advantage of this approach over conventional hypothesis tests is that prior knowledge can be incorporated to enhance predictive power. We demonstrate in a simulation study and on biological data, that integrating complementary information greatly improves prediction accuracy. To search for significant modules, we perform hierarchical clustering with multiscale bootstrap resampling. We demonstrate the power of the proposed methodologies in applications to Ewing's sarcoma and human adult stem cells using publicly available and custom generated data, respectively. In the former application, we identify a gene module including many confirmed and highly promising therapeutic targets. Genes in the module are also significantly overrepresented in signalling pathways that are known to be critical for proliferation of Ewing's sarcoma cells. In the latter application, we predict a functional network of chromatin factors controlling epidermal stem cell fate. Further examinations using ChIP-seq, ChIP-qPCR and RT-qPCR reveal that the basis of their genetic interactions may arise from transcriptional cross regulation. A Bioconductor package

Oxygen concentration modulates cellular senescence and autophagy in human trophoblast cells.

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Seno, Kotomi; Tanikawa, Nao; Takahashi, Hironori; Ohkuchi, Akihide; Suzuki, Hirotada; Matsubara, Shigeki; Iwata, Hisataka; Kuwayama, Takehito; Shirasuna, Koumei

2018-02-15

We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O 2 concentrations for 24 hours. We examined the extent of senescence caused using senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). Physiological normoxia (5% O 2 ) decreased SA-β-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O 2 . Pathophysiological hypoxia (1% O 2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-β-Gal-positive cells and SASP secretion) in O 2 -independent manner. We conclude that O 2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiparametric classification links tumor microenvironments with tumor cell phenotype.

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Bojana Gligorijevic

2014-11-01

Full Text Available While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which

Potential contribution of phenotypically modulated smooth muscle cells and related inflammation in the development of experimental obstructive pulmonary vasculopathy in rats.

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Shoichiro Otsuki

Full Text Available We tested the hypothesis that phenotypically modulated smooth muscle cells (SMCs and related inflammation are associated with the progression of experimental occlusive pulmonary vascular disease (PVD. Occlusive PVD was induced by combined exposure to a vascular endothelial growth factor receptor tyrosine kinase inhibitor Sugen 5416 and hypobaric hypoxia for 3 weeks in rats, which were then returned to ambient air. Hemodynamic, morphometric, and immunohistochemical studies, as well as gene expression analyses, were performed at 3, 5, 8, and 13 weeks after the initial treatment (n = 78. Experimental animals developed pulmonary hypertension and right ventricular hypertrophy, and exhibited a progressive increase in indices of PVD, including cellular intimal thickening and intimal fibrosis. Cellular intimal lesions comprised α smooth muscle actin (α SMA+, SM1+, SM2+/-, vimentin+ immature SMCs that were covered by endothelial monolayers, while fibrous intimal lesions typically included α SMA+, SM1+, SM2+, vimentin+/- mature SMCs. Plexiform lesions comprised α SMA+, vimentin+, SM1-, SM2- myofibroblasts covered by endothelial monolayers. Immature SMC-rich intimal and plexiform lesions were proliferative and were infiltrated by macrophages, while fibrous intimal lesions were characterized by lower proliferative abilities and were infiltrated by few macrophages. Compared with controls, the number of perivascular macrophages was already higher at 3 weeks and progressively increased during the experimental period; gene expression of pulmonary hypertension-related inflammatory molecules, including IL6, MCP1, MMP9, cathepsin-S, and RANTES, was persistently or progressively up-regulated in lungs of experimental animals. We concluded that phenotypically modulated SMCs and related inflammation are potentially associated with the progression of experimental obstructive PVD.

Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

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Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

1994-10-01

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils

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Galli, Stephen J; Borregaard, Niels; Wynn, Thomas A

2011-01-01

Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function......). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially...... and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents...

Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils

DEFF Research Database (Denmark)

Galli, Stephen J; Borregaard, Niels; Wynn, Thomas A

2011-01-01

). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially......Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function...... and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents...

Predicting biomaterial property-dendritic cell phenotype relationships from the multivariate analysis of responses to polymethacrylates

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Kou, Peng Meng; Pallassana, Narayanan; Bowden, Rebeca; Cunningham, Barry; Joy, Abraham; Kohn, Joachim; Babensee, Julia E.

2011-01-01

Dendritic cells (DCs) play a critical role in orchestrating the host responses to a wide variety of foreign antigens and are essential in maintaining immune tolerance. Distinct biomaterials have been shown to differentially affect the phenotype of DCs, which suggested that biomaterials may be used to modulate immune response towards the biologic component in combination products. The elucidation of biomaterial property-DC phenotype relationships is expected to inform rational design of immuno-modulatory biomaterials. In this study, DC response to a set of 12 polymethacrylates (pMAs) was assessed in terms of surface marker expression and cytokine profile. Principal component analysis (PCA) determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an immature DC phenotype. Partial square linear regression, a multivariate modeling approach, was implemented and successfully predicted biomaterial-induced DC phenotype in terms of surface marker expression from biomaterial properties with R2prediction = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R2prediction = 0.80. These results demonstrated that immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection. PMID:22136715

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

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Stephanie Plog

Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

NARCIS (Netherlands)

van Hemert, Saskia; Meijerink, Marjolein; Molenaar, Douwe; Bron, Peter A.; de Vos, Paul; Kleerebezem, Michiel; Wells, Jerry M.; Marco, Maria L.

2010-01-01

Background: Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic

BAG3 promotes the phenotypic transformation of primary rat vascular smooth muscle cells via TRAIL.

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Fu, Yao; Chang, Ye; Chen, Shuang; Li, Yuan; Chen, Yintao; Sun, Guozhe; Yu, Shasha; Ye, Ning; Li, Chao; Sun, Yingxian

2018-05-01

Under normal physiological condition, the mature vascular smooth muscle cells (VSMCs) show differentiated phenotype. In response to various environmental stimuluses, VSMCs convert from the differentiated phenotype to dedifferentiated phenotype characterized by the increased ability of proliferation/migration and the reduction of contractile ability. The phenotypic transformation of VSMCs played an important role in atherosclerosis. Both Bcl-2-associated athanogene 3 (BAG3) and tumor necrosis factor-related apopt-osis inducing ligand (TRAIL) involved in apoptosis. The relationship between BAG3 and TRAIL and their effects the proliferation and migration in VSMCs are rarely reported. This study investigated the effects of BAG3 on the phenotypic modulation and the potential underlying mechanisms in primary rat VSMCs. Primary rat VSMCs were extracted and cultured in vitro. Cell proliferation was detected by cell counting, real-time cell analyzer (RTCA) and EdU incorporation. Cell migration was detected by wound healing, Transwell and RTCA. BAG3 and TRAIL were detected using real-time PCR and western blotting and the secreted proteins in the cultured media by dot blot. The expression of BAG3 increased with continued passages in cultured primary VSMCs. BAG3 promoted the proliferation and migration of primary rat VSMC in a time-dependent manner. BAG3 significantly increased the expression of TRAIL while had no effects on its receptors. TRAIL knockdown or blocking by neutralizing antibody inhibited the proliferation of VSMCs induced by BAG3. TRAIL knockdown exerted no obvious influence on the migration of VSMCs. Based on this study, we report for the first time that BAG3 was expressed in cultured primary rat VSMCs and the expression of BAG3 increased with continued passages. Furthermore, BAG3 promoted the proliferation of VSMCs via increasing the expression of TRAIL. In addition, we also demonstrated that BAG3 promoted the migration of VSMCs independent of TRAIL

Aberrant phenotypes in peripheral T cell lymphomas.

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Hastrup, N; Ralfkiaer, E; Pallesen, G

1989-01-01

Seventy six peripheral T cell lymphomas were examined immunohistologically to test their reactivity with a panel of monoclonal antibodies against 11 T cell associated antigens (CD1-8, CD27, UCHL1, and the T cell antigen receptor). Sixty two (82%) lymphomas showed aberrant phenotypes, and four main categories were distinguished as follows: (i) lack of one or several pan-T cell antigens (49, 64% of the cases); (ii) loss of both the CD4 and CD8 antigens (11, 15% of the cases); (iii) coexpression of the CD4 and CD8 antigens (13, 17% of the cases); and (iv) expression of the CD1 antigen (eight, 11% of the cases). No correlation was seen between the occurrence of aberrant phenotypes and the histological subtype. It is concluded that the demonstration of an aberrant phenotype is a valuable supplement to histological assessment in the diagnosis of peripheral T cell lymphomas. It is recommended that the panel of monoclonal antibodies against T cell differentiation antigens should be fairly large, as apparently any antigen may be lost in the process of malignant transformation. Images Figure PMID:2469701

Structural phenotyping of stem cell-derived cardiomyocytes.

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Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

2015-03-10

Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Toward automatic phenotyping of retinal images from genetically determined mono- and dizygotic twins using amplitude modulation-frequency modulation methods

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Soliz, P.; Davis, B.; Murray, V.; Pattichis, M.; Barriga, S.; Russell, S.

2010-03-01

This paper presents an image processing technique for automatically categorize age-related macular degeneration (AMD) phenotypes from retinal images. Ultimately, an automated approach will be much more precise and consistent in phenotyping of retinal diseases, such as AMD. We have applied the automated phenotyping to retina images from a cohort of mono- and dizygotic twins. The application of this technology will allow one to perform more quantitative studies that will lead to a better understanding of the genetic and environmental factors associated with diseases such as AMD. A method for classifying retinal images based on features derived from the application of amplitude-modulation frequency-modulation (AM-FM) methods is presented. Retinal images from identical and fraternal twins who presented with AMD were processed to determine whether AM-FM could be used to differentiate between the two types of twins. Results of the automatic classifier agreed with the findings of other researchers in explaining the variation of the disease between the related twins. AM-FM features classified 72% of the twins correctly. Visual grading found that genetics could explain between 46% and 71% of the variance.

Phenotypic equilibrium as probabilistic convergence in multi-phenotype cell population dynamics.

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Da-Quan Jiang

Full Text Available We consider the cell population dynamics with n different phenotypes. Both the Markovian branching process model (stochastic model and the ordinary differential equation (ODE system model (deterministic model are presented, and exploited to investigate the dynamics of the phenotypic proportions. We will prove that in both models, these proportions will tend to constants regardless of initial population states ("phenotypic equilibrium" under weak conditions, which explains the experimental phenomenon in Gupta et al.'s paper. We also prove that Gupta et al.'s explanation is the ODE model under a special assumption. As an application, we will give sufficient and necessary conditions under which the proportion of one phenotype tends to 0 (die out or 1 (dominate. We also extend our results to non-Markovian cases.

Incorporation of Biomaterials in Multicellular Aggregates Modulates Pluripotent Stem Cell Differentiation

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Bratt-Leal, Andrés M.; Carpenedo, Richard L.; Ungrin, Mark; Zandstra, Peter W.; McDevitt, Todd C.

2010-01-01

Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes. PMID:20864164

High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.

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Czerniecki, Stefan M; Cruz, Nelly M; Harder, Jennifer L; Menon, Rajasree; Annis, James; Otto, Edgar A; Gulieva, Ramila E; Islas, Laura V; Kim, Yong Kyun; Tran, Linh M; Martins, Timothy J; Pippin, Jeffrey W; Fu, Hongxia; Kretzler, Matthias; Shankland, Stuart J; Himmelfarb, Jonathan; Moon, Randall T; Paragas, Neal; Freedman, Benjamin S

2018-05-15

Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening. Copyright © 2018 Elsevier Inc. All rights reserved.

HTLV-1 modulates the frequency and phenotype of FoxP3+CD4+ T cells in virus-infected individuals

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Satou Yorifumi

2012-05-01

Full Text Available Abstract Background HTLV-1 utilizes CD4 T cells as the main host cell and maintains the proviral load via clonal proliferation of infected CD4+ T cells. Infection of CD4+ T cells by HTLV-1 is therefore thought to play a pivotal role in HTLV-1-related pathogenicity, including leukemia/lymphoma of CD4+ T cells and chronic inflammatory diseases. Recently, it has been reported that a proportion of HTLV-1 infected CD4+ T cells express FoxP3, a master molecule of regulatory T cells. However, crucial questions remain unanswered on the relationship between HTLV-1 infection and FoxP3 expression. Results To investigate the effect of HTLV-1 infection on CD4+ T-cell subsets, we used flow cytometry to analyze the T-cell phenotype and HTLV-1 infection in peripheral mononuclear cells (PBMCs of four groups of subjects, including 23 HTLV-1-infected asymptomatic carriers (AC, 10 patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP, 10 patients with adult T-cell leukemia (ATL, and 10 healthy donors. The frequency of FoxP3+ cells in CD4+ T cells in AC with high proviral load and patients with HAM/TSP or ATL was higher than that in uninfected individuals. The proviral load was positively correlated with the percentage of CD4+ T cells that were FoxP3+. The CD4+FoxP3+ T cells, themselves, were frequently infected with HTLV-1. We conclude that FoxP3+ T- cells are disproportionately infected with HTLV-1 during chronic infection. We next focused on PBMCs of HAM/TSP patients. The expression levels of the Treg associated molecules CTLA-4 and GITR were decreased in CD4+FoxP3+ T cells. Further we characterized FoxP3+CD4+ T-cell subsets by staining CD45RA and FoxP3, which revealed an increase in CD45RA−FoxP3low non-suppressive T-cells. These findings can reconcile the inflammatory phenotype of HAM/TSP with the observed increase in frequency of FoxP3+ cells. Finally, we analyzed ATL cells and observed not only a high frequency of FoxP3 expression

Reciprocal modulation of mesenchymal stem cells and tumor cells promotes lung cancer metastasis

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Giulia Fregni

2018-03-01

Full Text Available Metastasis is a multi-step process in which direct crosstalk between cancer cells and their microenvironment plays a key role. Here, we assessed the effect of paired tumor-associated and normal lung tissue mesenchymal stem cells (MSCs on the growth and dissemination of primary human lung carcinoma cells isolated from the same patients. We show that the tumor microenvironment modulates MSC gene expression and identify a four-gene MSC signature that is functionally implicated in promoting metastasis. We also demonstrate that tumor-associated MSCs induce the expression of genes associated with an aggressive phenotype in primary lung cancer cells and selectively promote their dissemination rather than local growth. Our observations provide insight into mechanisms by which the stroma promotes lung cancer metastasis. Keywords: Tumor-associated MSCs, lung cancer, metastasis, GREM1, LOXL2, ADAMTS12, ITGA11

IGF-1 Has Plaque-Stabilizing Effects in Atherosclerosis by Altering Vascular Smooth Muscle Cell Phenotype

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von der Thüsen, Jan H.; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J.C.; Biessen, Erik A.L.

2011-01-01

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. PMID:21281823

Induction of appropriate Th-cell phenotypes: cellular decision-making in heterogeneous environments.

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van den Ham, H-J; Andeweg, A C; de Boer, R J

2013-11-01

Helper T (Th)-cell differentiation is a key event in the development of the adaptive immune response. By the production of a range of cytokines, Th cells determine the type of immune response that is raised against an invading pathogen. Th cells can adopt many different phenotypes, and Th-cell phenotype decision-making is crucial in mounting effective host responses. This review discusses the different Th-cell phenotypes that have been identified and how Th cells adopt a particular phenotype. The regulation of Th-cell phenotypes has been studied extensively using mathematical models, which have explored the role of regulatory mechanisms such as autocrine cytokine signalling and cross-inhibition between self-activating transcription factors. At the single cell level, Th responses tend to be heterogeneous, but corrections can be made soon after T-cell activation. Although pathogens and the innate immune system provide signals that direct the induction of Th-cell phenotypes, these instructive mechanisms could be easily subverted by pathogens. We discuss that a model of success-driven feedback would select the most appropriate phenotype for clearing a pathogen. Given the heterogeneity in the induction phase of the Th response, such a success-driven feedback loop would allow the selection of effective Th-cell phenotypes while terminating incorrect responses. © 2013 John Wiley & Sons Ltd.

The Chado Natural Diversity module: a new generic database schema for large-scale phenotyping and genotyping data.

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Jung, Sook; Menda, Naama; Redmond, Seth; Buels, Robert M; Friesen, Maren; Bendana, Yuri; Sanderson, Lacey-Anne; Lapp, Hilmar; Lee, Taein; MacCallum, Bob; Bett, Kirstin E; Cain, Scott; Clements, Dave; Mueller, Lukas A; Main, Dorrie

2011-01-01

Linking phenotypic with genotypic diversity has become a major requirement for basic and applied genome-centric biological research. To meet this need, a comprehensive database backend for efficiently storing, querying and analyzing large experimental data sets is necessary. Chado, a generic, modular, community-based database schema is widely used in the biological community to store information associated with genome sequence data. To meet the need to also accommodate large-scale phenotyping and genotyping projects, a new Chado module called Natural Diversity has been developed. The module strictly adheres to the Chado remit of being generic and ontology driven. The flexibility of the new module is demonstrated in its capacity to store any type of experiment that either uses or generates specimens or stock organisms. Experiments may be grouped or structured hierarchically, whereas any kind of biological entity can be stored as the observed unit, from a specimen to be used in genotyping or phenotyping experiments, to a group of species collected in the field that will undergo further lab analysis. We describe details of the Natural Diversity module, including the design approach, the relational schema and use cases implemented in several databases.

Depletion of tumor-associated macrophages switches the epigenetic profile of pancreatic cancer infiltrating T cells and restores their anti-tumor phenotype.

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Borgoni, Simone; Iannello, Andrea; Cutrupi, Santina; Allavena, Paola; D'Incalci, Maurizio; Novelli, Francesco; Cappello, Paola

2018-01-01

Pancreatic Ductal Adenocarcinoma (PDA) is characterized by a complex tumor microenvironment that supports its progression, aggressiveness and resistance to therapies. The delicate interplay between cancer and immune cells creates the conditions for PDA development, particularly due to the functional suppression of T cell anti-tumor effector activity. However, some of the mechanisms involved in this process are still poorly understood. In this study, we analyze whether the functional and epigenetic profile of T cells that infiltrate PDA is modulated by the microenvironment, and in particular by tumor-associated macrophages (TAMs). CD4 and CD8 T cells obtained from mice orthotopically injected with syngeneic PDA cells, and untreated or treated with Trabectedin, a cytotoxic drug that specifically targets TAMs, were sorted and analyzed by flow cytometry and characterized for their epigenetic profile. Assessment of cytokine production and the epigenetic profile of genes coding for IL10, T-bet and PD1 revealed that T cells that infiltrated PDA displayed activated Il10 promoter and repressed T-bet activity, in agreement with their regulatory phenotype (IL10 high /IFNγ low , PD1 high ). By contrast, in Trabectedin-treated mice, PDA-infiltrating T cells displayed repressed Il10 and Pdcd1 and activated T-bet promoter activity, in accordance with their anti-tumor effector phenotype (IL10 low /IFNγ high ), indicating a key role of TAMs in orchestrating functions of PDA-infiltrating T cells by modulating their epigenetic profile towards a pro-tumoral phenotype. These results suggest the targeting of TAMs as an efficient strategy to obtain an appropriate T cell anti-tumor immune response and open new potential combinations for PDA treatment.

Heterogeneity of functional properties of Clone 66 murine breast cancer cells expressing various stem cell phenotypes.

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Mukhopadhyay, Partha; Farrell, Tracy; Sharma, Gayatri; McGuire, Timothy R; O'Kane, Barbara; Sharp, J Graham

2013-01-01

Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. The proportion of cells expressing CD44(high)CD24(low/neg), side population (SP) cells, ALDH1(+), CD49f(high), CD133(high), and CD34(high) differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1(+), CD34(low), and CD49f(high) suggested properties of transit amplifying cells. Colony formation was higher from ALDH1(-) and non-SP cells than ALDH1(+) and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than "non-stem" cells. Fewer SP cells were needed to form tumors than ALDH1(+) cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

Modulation of invasive phenotype by interstitial pressure-driven convection in aggregates of human breast cancer cells.

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Joe Tien

Full Text Available This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion.

Discrimination of meniscal cell phenotypes using gene expression profiles

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M Son

2012-03-01

Full Text Available The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus. The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution.

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Stepanenko, A A; Dmitrenko, V V

2015-09-15

293 cell line (widely known as the Human Embryonic Kidney 293 cells) and its derivatives were the most used cells after HeLa in cell biology studies and after CHO in biotechnology as a vehicle for the production of adenoviral vaccines and recombinant proteins, for analysis of the neuronal synapse formation, in electrophysiology and neuropharmacology. Despite the historically long-term productive exploitation, the origin, phenotype, karyotype, and tumorigenicity of 293 cells are still debated. 293 cells were considered the kidney epithelial cells or even fibroblasts. However, 293 cells demonstrate no evident tissue-specific gene expression signature and express the markers of renal progenitor cells, neuronal cells and adrenal gland. This complicates efforts to reveal the authentic cell type/tissue of origin. On the other hand, the potential to propagate the highly neurotropic viruses, inducible synaptogenesis, functionality of the endogenous neuron-specific voltage-gated channels, and response to the diverse agonists implicated in neuronal signaling give credibility to consider 293 cells of neuronal lineage phenotype. The compound phenotype of 293 cells can be due to heterogeneous, unstable karyotype. The mean chromosome number and chromosome aberrations differ between 293 cells and derivatives as well as between 293 cells from the different cell banks/labs. 293 cells are tumorigenic, whereas acute changes of expression of the cancer-associated genes aggravate tumorigenicity by promoting chromosome instability. Importantly, the procedure of a stable empty vector transfection can also impact karyotype and phenotype. The discussed issues caution against misinterpretations and pitfalls during the different experimental manipulations with 293 cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Socio-Cognitive Phenotypes Differentially Modulate Large-Scale Structural Covariance Networks.

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Valk, Sofie L; Bernhardt, Boris C; Böckler, Anne; Trautwein, Fynn-Mathis; Kanske, Philipp; Singer, Tania

2017-02-01

Functional neuroimaging studies have suggested the existence of 2 largely distinct social cognition networks, one for theory of mind (taking others' cognitive perspective) and another for empathy (sharing others' affective states). To address whether these networks can also be dissociated at the level of brain structure, we combined behavioral phenotyping across multiple socio-cognitive tasks with 3-Tesla MRI cortical thickness and structural covariance analysis in 270 healthy adults, recruited across 2 sites. Regional thickness mapping only provided partial support for divergent substrates, highlighting that individual differences in empathy relate to left insular-opercular thickness while no correlation between thickness and mentalizing scores was found. Conversely, structural covariance analysis showed clearly divergent network modulations by socio-cognitive and -affective phenotypes. Specifically, individual differences in theory of mind related to structural integration between temporo-parietal and dorsomedial prefrontal regions while empathy modulated the strength of dorsal anterior insula networks. Findings were robust across both recruitment sites, suggesting generalizability. At the level of structural network embedding, our study provides a double dissociation between empathy and mentalizing. Moreover, our findings suggest that structural substrates of higher-order social cognition are reflected rather in interregional networks than in the the local anatomical markup of specific regions per se. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cell Phenotype Transitions in Cardiovascular Calcification

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Luis Hortells

2018-03-01

Full Text Available Cardiovascular calcification was originally considered a passive, degenerative process, however with the advance of cellular and molecular biology techniques it is now appreciated that ectopic calcification is an active biological process. Vascular calcification is the most common form of ectopic calcification, and aging as well as specific disease states such as atherosclerosis, diabetes, and genetic mutations, exhibit this pathology. In the vessels and valves, endothelial cells, smooth muscle cells, and fibroblast-like cells contribute to the formation of extracellular calcified nodules. Research suggests that these vascular cells undergo a phenotypic switch whereby they acquire osteoblast-like characteristics, however the mechanisms driving the early aspects of these cell transitions are not fully understood. Osteoblasts are true bone-forming cells and differentiate from their pluripotent precursor, the mesenchymal stem cell (MSC; vascular cells that acquire the ability to calcify share aspects of the transcriptional programs exhibited by MSCs differentiating into osteoblasts. What is unknown is whether a fully-differentiated vascular cell directly acquires the ability to calcify by the upregulation of osteogenic genes or, whether these vascular cells first de-differentiate into an MSC-like state before obtaining a “second hit” that induces them to re-differentiate down an osteogenic lineage. Addressing these questions will enable progress in preventative and regenerative medicine strategies to combat vascular calcification pathologies. In this review, we will summarize what is known about the phenotypic switching of vascular endothelial, smooth muscle, and valvular cells.

Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

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Mulvey, Hillary E.; Chang, Audrey; Adler, Jason; Del Tatto, Michael; Perez, Kimberly; Quesenberry, Peter J.; Chatterjee, Devasis

2015-01-01

Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer

Self-glycolipids modulate dendritic cells changing the cytokine profiles of committed autoreactive T cells.

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Karsten Buschard

Full Text Available The impact of glycolipids of non-mammalian origin on autoimmune inflammation has become widely recognized. Here we report that the naturally occurring mammalian glycolipids, sulfatide and β-GalCer, affect the differentiation and the quality of antigen presentation by monocyte-derived dendritic cells (DCs. In response to sulfatide and β-GalCer, monocytes develop into immature DCs with higher expression of HLA-DR and CD86 but lower expression of CD80, CD40 and CD1a and lower production of IL-12 compared to non-modulated DCs. Self-glycolipid-modulated DCs responded to lipopolysaccharide (LPS by changing phenotype but preserved low IL-12 production. Sulfatide, in particular, reduced the capacity of DCs to stimulate autoreactive Glutamic Acid Decarboxylase (GAD65 - specific T cell response and promoted IL-10 production by the GAD65-specific clone. Since sulfatide and β-GalCer induced toll-like receptor (TLR-mediated signaling, we hypothesize that self-glycolipids deliver a (tolerogenic polarizing signal to differentiating DCs, facilitating the maintenance of self-tolerance under proinflammatory conditions.

In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype.

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Walker, J L; Bleaken, B M; Romisher, A R; Alnwibit, A A; Menko, A S

2018-05-02

Following injury, mesenchymal repair cells are activated to function as leader cells that modulate wound healing. These cells have the potential to differentiate to myofibroblasts, resulting in fibrosis and scarring. The signals underlying these differing pathways are complex and incompletely understood. The ex vivo mock cataract surgery cultures are an attractive model with which to address this question. With this model we study, concurrently, the mechanisms that control mesenchymal leader cell function in injury repair within their native microenvironment, and the signals that induce this same cell population to acquire a myofibroblast phenotype when these cells encounter the environment of the adjacent tissue culture platform. Here, we show that upon injury, the cytoskeletal protein vimentin is released into the extracellular space, binds to the cell surface of the mesenchymal leader cells located at the wound edge in the native matrix environment, and supports wound closure. In pro-fibrotic environments, the extracellular vimentin pool also links specifically to the mesenchymal leader cells, and has an essential role in signaling their fate change to a myofibroblast. These findings suggest a novel role for extracellular, cell-surface-associated vimentin in mediating repair-cell function in wound repair and in transitioning these cells to a myofibroblast phenotype. Movie S1 Movie S1 Collective movement of mesenchymal leader and epithelial follower cells across the tissue culture substrate (ECZ) in response to injury was followed by time-lapse imaging from D0-D3. The mesenchymal cells at the leading edge were easily distinguished morphologically from the lens epithelial follower cells.

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells

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Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M.; Wells, Alan

2016-01-01

Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

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Hatta, Mitsutoki; Naganuma, Kaori; Kato, Kenichi; Yamazaki, Jun

2015-01-01

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

Energy Technology Data Exchange (ETDEWEB)

Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan); Naganuma, Kaori [Department of Oral and Maxillofacial Surgery, Fukuoka Dental College, Fukuoka (Japan); Kato, Kenichi; Yamazaki, Jun [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan)

2015-12-04

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

Photovoltaic cell module and method of forming

Science.gov (United States)

Howell, Malinda; Juen, Donnie; Ketola, Barry; Tomalia, Mary Kay

2017-12-12

A photovoltaic cell module, a photovoltaic array including at least two modules, and a method of forming the module are provided. The module includes a first outermost layer and a photovoltaic cell disposed on the first outermost layer. The module also includes a second outermost layer disposed on the photovoltaic cell and sandwiching the photovoltaic cell between the second outermost layer and the first outermost layer. The method of forming the module includes the steps of disposing the photovoltaic cell on the first outermost layer, disposing a silicone composition on the photovoltaic cell, and compressing the first outermost layer, the photovoltaic cell, and the second layer to form the photovoltaic cell module.

MicroRNA-155 Modulates Acute Graft-versus-Host Disease by Impacting T Cell Expansion, Migration, and Effector Function.

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Zitzer, Nina C; Snyder, Katiri; Meng, Xiamoei; Taylor, Patricia A; Efebera, Yvonne A; Devine, Steven M; Blazar, Bruce R; Garzon, Ramiro; Ranganathan, Parvathi

2018-06-15

MicroRNA-155 (miR-155) is a small noncoding RNA critical for the regulation of inflammation as well as innate and adaptive immune responses. MiR-155 has been shown to be dysregulated in both donor and recipient immune cells during acute graft-versus-host disease (aGVHD). We previously reported that miR-155 is upregulated in donor T cells of mice and humans with aGVHD and that mice receiving miR-155-deficient (miR155 -/- ) splenocytes had markedly reduced aGVHD. However, molecular mechanisms by which miR-155 modulates T cell function in aGVHD have not been fully investigated. We identify that miR-155 expression in both donor CD8 + T cells and conventional CD4 + CD25 - T cells is pivotal for aGVHD pathogenesis. Using murine aGVHD transplant experiments, we show that miR-155 strongly impacts alloreactive T cell expansion through multiple distinct mechanisms, modulating proliferation in CD8 + donor T cells and promoting exhaustion in donor CD4 + T cells in both the spleen and colon. Additionally, miR-155 drives a proinflammatory Th1 phenotype in donor T cells in these two sites, and miR-155 -/- donor T cells are polarized toward an IL-4-producing Th2 phenotype. We further demonstrate that miR-155 expression in donor T cells regulates CCR5 and CXCR4 chemokine-dependent migration. Notably, we show that miR-155 expression is crucial for donor T cell infiltration into multiple target organs. These findings provide further understanding of the role of miR-155 in modulating aGVHD through T cell expansion, effector cytokine production, and migration. Copyright © 2018 by The American Association of Immunologists, Inc.

Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells

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Hamada, Shin; Masamune, Atsushi; Takikawa, Tetsuya; Suzuki, Noriaki; Kikuta, Kazuhiro; Hirota, Morihisa; Hamada, Hirofumi; Kobune, Masayoshi; Satoh, Kennichi; Shimosegawa, Tooru

2012-01-01

Highlights: ► Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. ► Pancreatic cancer cells co-cultured with PSCs showed enhanced spheroid formation. ► Expression of stem cell-related genes ABCG2, Nestin and LIN28 was increased. ► Co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. ► This study suggested a novel role of PSCs as a part of the cancer stem cell niche. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Recent studies have identified that a portion of cancer cells, called “cancer stem cells”, within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the “stemness” of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

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Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

[Notochord cells enhance proliferation and phenotype-keeping of intervertebral disc chondroid cells].

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Zhao, Xianfeng; Liu, Hao; Feng, Ganjun; Deng, Li; Li, Xiuqun; Liang, Tao

2008-08-01

To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to evaluate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell proliferation and phenotype. The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) and co-cultured with notochord cells (group B) (1:1), and cell proliferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 microm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 microm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P > 0.05). The co-cultured cells (group B) increased in cell proliferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell proliferation in group B was more than that in group A after 4-day culture (P notochord cells are conducive for the proliferation and phenotype-keeping of the chondroid cells and

A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

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Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

2018-06-11

We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

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Hisataka Ogawa

Full Text Available Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

Innate immunity phenotypic features point toward simultaneous raise of activation and modulation events following 17DD live attenuated yellow fever first-time vaccination.

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Martins, Marina Angela; Silva, Maria Luiza; Elói-Santos, Silvana Maria; Ribeiro, José Geraldo Leite; Peruhype-Magalhães, Vanessa; Marciano, Ana Paula Vieira; Homma, Akira; Kroon, Erna Geessien; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis

2008-02-26

Detailed multiparametric phenotypic investigation aiming to characterize the kinetics of the innate immune response in the peripheral blood following 17DD yellow fever (17DD-YF) first-time vaccination was performed. Results showed increased frequency of monocytes and NK cell subpopulations besides unexpected up-regulation of granulocytes activation status (CD28+/CD23+ and CD28+/HLA-DR+, respectively). Up-regulation of Fcgamma-R and IL-10-R expression emerge as putative events underlying the mixed pattern of phenotypic features triggered by the 17DD yellow fever (17DD-YF) vaccination. Mixed pattern of chemokine receptors expression further support our hypothesis that a parallel establishment of activation/modulation microenvironment plays a pivotal role in the protective immunity triggered by the 17DD-YF vaccine.

Loss of end-differentiated β-cell phenotype following pancreatic islet transplantation.

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Anderson, S J; White, M G; Armour, S L; Maheshwari, R; Tiniakos, D; Muller, Y D; Berishvili, E; Berney, T; Shaw, J A M

2018-03-01

Replacement of pancreatic β-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced β-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated β-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin + /urocortin-3 - cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype

Science.gov (United States)

Gabrusiewicz, Konrad; Rodriguez, Benjamin; Wei, Jun; Hashimoto, Yuuri; Healy, Luke M.; Maiti, Sourindra N.; Wang, Qianghu; Elakkad, Ahmed; Liebelt, Brandon D.; Yaghi, Nasser K.; Ezhilarasan, Ravesanker; Huang, Neal; Weinberg, Jeffrey S.; Prabhu, Sujit S.; Rao, Ganesh; Sawaya, Raymond; Langford, Lauren A.; Bruner, Janet M.; Fuller, Gregory N.; Bar-Or, Amit; Li, Wei; Colen, Rivka R.; Curran, Michael A.; Bhat, Krishna P.; Antel, Jack P.; Cooper, Laurence J.; Sulman, Erik P.; Heimberger, Amy B.

2016-01-01

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages. PMID:26973881

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models

International Nuclear Information System (INIS)

Phelan, J. P.; Reen, F. J.; Dunphy, N.; O'Connor, R.; O'Gara, F.

2016-01-01

The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression

Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

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Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

2009-11-01

Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

Alterations in Mesenteric Lymph Node T Cell Phenotype and Cytokine Secretion are Associated with Changes in Thymocyte Phenotype after LP-BM5 Retrovirus Infection

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Maria C. Lopez

2005-01-01

Full Text Available In this study, mouse MLN cells and thymocytes from advanced stages of LP-BM5 retrovirus infection were studied. A decrease in the percentage of IL-7+ cells and an increase in the percentage of IL-16+ cells in the MLN indicated that secretion of these cytokines was also altered after LP-BM5 infection. The percentage of MLN T cells expressing IL-7 receptors was significantly reduced, while the percentage of MLN T cells expressing TNFR-p75 and of B cells expressing TNFR-p55 increased. Simultaneous analysis of surface markers and cytokine secretion was done in an attempt to understand whether the deregulation of IFN-Υ secretion could be ascribed to a defined cell phenotype, concluding that all T cell subsets studied increased IFN-Υ secretion after retrovirus infection. Finally, thymocyte phenotype was further analyzed trying to correlate changes in thymocyte phenotype with MLN cell phenotype. The results indicated that the increase in single positive either CD4+CD8- or CD4- CD8+ cells was due to accumulation of both immature (CD3- and mature (CD3+ single positive thymocytes. Moreover, single positive mature thymocytes presented a phenotype similar to the phenotype previously seen on MLN T cells. In summary, we can conclude that LP-BM5 uses the immune system to reach the thymus where it interferes with the generation of functionally mature T cells, favoring the development of T cells with an abnormal phenotype. These new T cells are activated to secrete several cytokines that in turn will favor retrovirus replication and inhibit any attempt of the immune system to control infection.

Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

Science.gov (United States)

de Souza, Alessandra A; Ionescu, Michael; Baccari, Clelia; da Silva, Aline M; Lindow, Steven E

2013-06-01

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

Expression of the C-terminal family 22 carbohydratebinding module ...

African Journals Online (AJOL)

... not reveal marked cell wall phenotype. In addition, there were no observable changes in the height or the appearance of the transgenic plants expressing the CBM22-2 module. The results indicate that the family 22 carbohydrate binding module is not a potential candidate for use in in planta modification of the cell wall.

Smooth muscle cell phenotypic switching in stroke.

Science.gov (United States)

Poittevin, Marine; Lozeron, Pierre; Hilal, Rose; Levy, Bernard I; Merkulova-Rainon, Tatiana; Kubis, Nathalie

2014-06-01

Disruption of cerebral blood flow after stroke induces cerebral tissue injury through multiple mechanisms that are not yet fully understood. Smooth muscle cells (SMCs) in blood vessel walls play a key role in cerebral blood flow control. Cerebral ischemia triggers these cells to switch to a phenotype that will be either detrimental or beneficial to brain repair. Moreover, SMC can be primarily affected genetically or by toxic metabolic molecules. After stroke, this pathological phenotype has an impact on the incidence, pattern, severity, and outcome of the cerebral ischemic disease. Although little research has been conducted on the pathological role and molecular mechanisms of SMC in cerebrovascular ischemic diseases, some therapeutic targets have already been identified and could be considered for further pharmacological development. We examine these different aspects in this review.

Dickkopf-3, a tissue-derived modulator of local T cell responses

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Michael eMeister

2015-02-01

Full Text Available The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T cell reactivity. Dkk3 is a secreted, mainly tissue derived protein with highest expression in organs considered as immune privileged such as the eye, embryo, placenta and brain. While T cell development and activation status in naïve Dkk3 deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T cell mediated rejection. Moreover, genetic deletion or antibody mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE. This phenotype was accompanied by a change of T cell polarization displayed by an increase of IFNγ producing T cells within in the CNS. In the wild type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4+ and CD8+ T cell responses.

Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity.

Science.gov (United States)

Binder, Dennis; Drepper, Thomas; Jaeger, Karl-Erich; Delvigne, Frank; Wiechert, Wolfgang; Kohlheyer, Dietrich; Grünberger, Alexander

2017-07-01

In natural habitats, microbes form multispecies communities that commonly face rapidly changing and highly competitive environments. Thus, phenotypic heterogeneity has evolved as an innate and important survival strategy to gain an overall fitness advantage over cohabiting competitors. However, in defined artificial environments such as monocultures in small- to large-scale bioreactors, cell-to-cell variations are presumed to cause reduced production yields as well as process instability. Hence, engineering microbial production toward phenotypic homogeneity is a highly promising approach for synthetic biology and bioprocess optimization. In this review, we discuss recent studies that have unraveled the cell-to-cell heterogeneity observed during bacterial gene expression and metabolite production as well as the molecular mechanisms involved. In addition, current single-cell technologies are briefly reviewed with respect to their applicability in exploring cell-to-cell variations. We highlight emerging strategies and tools to reduce phenotypic heterogeneity in biotechnological expression setups. Here, strain or inducer modifications are combined with cell physiology manipulations to achieve the ultimate goal of equalizing bacterial populations. In this way, the majority of cells can be forced into high productivity, thus reducing less productive subpopulations that tend to consume valuable resources during production. Modifications in uptake systems, inducer molecules or nutrients represent valuable tools for diminishing heterogeneity. Finally, we address the challenge of transferring homogeneously responding cells into large-scale bioprocesses. Environmental heterogeneity originating from extrinsic factors such as stirring speed and pH, oxygen, temperature or nutrient distribution can significantly influence cellular physiology. We conclude that engineering microbial populations toward phenotypic homogeneity is an increasingly important task to take biotechnological

Modulation of the Senescence-Associated Inflammatory Phenotype in Human Fibroblasts by Olive Phenols

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Beatrice Menicacci

2017-10-01

Full Text Available Senescent cells display an increase in the secretion of growth factors, inflammatory cytokines and proteolytic enzymes, termed the “senescence-associated-secretory-phenotype” (SASP, playing a major role in many age-related diseases. The phenolic compounds present in extra-virgin olive oil are inhibitors of oxidative damage and have been reported to play a protective role in inflammation-related diseases. Particularly, hydroxytyrosol and oleuropein are the most abundant and more extensively studied. Pre-senescent human lung (MRC5 and neonatal human dermal (NHDF fibroblasts were used as cellular model to evaluate the effect of chronic (4–6 weeks treatment with 1 μM hydroxytyrosol (HT or 10 μM oleuropein aglycone (OLE on senescence/inflammation markers. Both phenols were effective in reducing β-galactosidase-positive cell number and p16 protein expression. In addition, senescence/inflammation markers such as IL-6 and metalloprotease secretion, and Ciclooxigenase type 2 (COX-2 and α-smooth-actin levels were reduced by phenol treatments. In NHDF, COX-2 expression, Nuclear Factor κ-light-chain-enhancer of activated B cells (NFκB protein level and nuclear localization were augmented with culture senescence and decreased by OLE and HT treatment. Furthermore, the inflammatory effect of Tumor Necrosis Factor α (TNFα exposure was almost completely abolished in OLE- and HT-pre-treated NHDF. Thus, the modulation of the senescence-associated inflammatory phenotype might be an important mechanism underlying the beneficial effects of olive oil phenols.

Cell shunt resistance and photovoltaic module performance

Energy Technology Data Exchange (ETDEWEB)

McMahon, T.J.; Basso, T.S.; Rummel, S.R. [National Renewable Energy Lab., Golden, CO (United States)

1996-05-01

Shunt resistance of cells in photovoltaic modules can affect module power output and could indicate flawed manufacturing processes and reliability problems. The authors describe a two-terminal diagnostic method to directly measure the shunt resistance of individual cells in a series-connected module non-intrusively, without deencapsulation. Peak power efficiency vs. light intensity was measured on a 12-cell, series-connected, single crystalline module having relatively high cell shunt resistances. The module was remeasured with 0.5-, 1-, and 2-ohm resistors attached across each cell to simulate shunt resistances of several emerging technologies. Peak power efficiencies decreased dramatically at lower light levels. Using the PSpice circuit simulator, the authors verified that cell shunt and series resistances can indeed be responsible for the observed peak power efficiency vs. intensity behavior. The authors discuss the effect of basic cell diode parameters, i.e., shunt resistance, series resistance, and recombination losses, on PV module performance as a function of light intensity.

Characterizing visible and invisible cell wall mutant phenotypes

Energy Technology Data Exchange (ETDEWEB)

Carpita, Nicholas C.; McCann, Maureen C.

2015-04-06

About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

Regulatory T cells expanded from HIV-1-infected individuals maintain phenotype, TCR repertoire and suppressive capacity.

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Mathieu Angin

Full Text Available While modulation of regulatory T cell (Treg function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4(+ Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region, characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

Migration Phenotype of Brain-Cancer Cells Predicts Patient Outcomes

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Chris L. Smith

2016-06-01

Full Text Available Glioblastoma multiforme is a heterogeneous and infiltrative cancer with dismal prognosis. Studying the migratory behavior of tumor-derived cell populations can be informative, but it places a high premium on the precision of in vitro methods and the relevance of in vivo conditions. In particular, the analysis of 2D cell migration may not reflect invasion into 3D extracellular matrices in vivo. Here, we describe a method that allows time-resolved studies of primary cell migration with single-cell resolution on a fibrillar surface that closely mimics in vivo 3D migration. We used this platform to screen 14 patient-derived glioblastoma samples. We observed that the migratory phenotype of a subset of cells in response to platelet-derived growth factor was highly predictive of tumor location and recurrence in the clinic. Therefore, migratory phenotypic classifiers analyzed at the single-cell level in a patient-specific way can provide high diagnostic and prognostic value for invasive cancers.

Tumor cell phenotype is sustained by selective MAPK oxidation in mitochondria.

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Soledad Galli

2008-06-01

Full Text Available Mitochondria are major cellular sources of hydrogen peroxide (H(2O(2, the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2O(2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2O(2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2O(2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2O(2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2O(2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei, the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2O(2 level. Like this, high H(2O(2 or directed mutation of redox-sensitive ERK2 Cys(214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to

Phenotype Clustering of Breast Epithelial Cells in Confocal Imagesbased on Nuclear Protein Distribution Analysis

Energy Technology Data Exchange (ETDEWEB)

Long, Fuhui; Peng, Hanchuan; Sudar, Damir; Levievre, Sophie A.; Knowles, David W.

2006-09-05

Background: The distribution of the chromatin-associatedproteins plays a key role in directing nuclear function. Previously, wedeveloped an image-based method to quantify the nuclear distributions ofproteins and showed that these distributions depended on the phenotype ofhuman mammary epithelial cells. Here we describe a method that creates ahierarchical tree of the given cell phenotypes and calculates thestatistical significance between them, based on the clustering analysisof nuclear protein distributions. Results: Nuclear distributions ofnuclear mitotic apparatus protein were previously obtained fornon-neoplastic S1 and malignant T4-2 human mammary epithelial cellscultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 andthe number of days in cultured. A probabilistic ensemble approach wasused to define a set of consensus clusters from the results of multipletraditional cluster analysis techniques applied to the nucleardistribution data. Cluster histograms were constructed to show how cellsin any one phenotype were distributed across the consensus clusters.Grouping various phenotypes allowed us to build phenotype trees andcalculate the statistical difference between each group. The resultsshowed that non-neoplastic S1 cells could be distinguished from malignantT4-2 cells with 94.19 percent accuracy; that proliferating S1 cells couldbe distinguished from differentiated S1 cells with 92.86 percentaccuracy; and showed no significant difference between the variousphenotypes of T4-2 cells corresponding to increasing tumor sizes.Conclusion: This work presents a cluster analysis method that canidentify significant cell phenotypes, based on the nuclear distributionof specific proteins, with high accuracy.

Phenotypic instability of Saos-2 cells in long-term culture

International Nuclear Information System (INIS)

Hausser, Heinz-Juergen; Brenner, Rolf E.

2005-01-01

The human osteosarcoma cell line Saos-2 is widely used as a model system for human osteoblastic cells, though its phenotypic stability has not been ascertained. We therefore propagated these cells over 100 passages and compared relevant phenotypic properties. In general, higher passage cells exhibited higher proliferation rates and lower specific alkaline phosphatase activities, though mineralization was significantly more pronounced in cultures of late passage cells. Whereas expression of most genes investigated did not vary profoundly, some genes exhibited remarkable differences. Decorin, for instance, that has been discussed as a regulator of proliferation and mineralization, is strongly expressed only in early passage cells, and two receptors for pleiotrophin and midkine exhibited an almost mutually exclusive expression pattern in early and late passage cells, respectively. Our observations indicate that special care is required when results obtained with Saos-2 cells with different culture history are to be compared

The origin of human complex diversity: Stochastic epistatic modules and the intrinsic compatibility between distributional robustness and phenotypic changeability.

Science.gov (United States)

Ijichi, Shinji; Ijichi, Naomi; Ijichi, Yukina; Imamura, Chikako; Sameshima, Hisami; Kawaike, Yoichi; Morioka, Hirofumi

2018-01-01

The continuing prevalence of a highly heritable and hypo-reproductive extreme tail of a human neurobehavioral quantitative diversity suggests the possibility that the reproductive majority retains the genetic mechanism for the extremes. From the perspective of stochastic epistasis, the effect of an epistatic modifier variant can randomly vary in both phenotypic value and effect direction among the careers depending on the genetic individuality, and the modifier careers are ubiquitous in the population distribution. The neutrality of the mean genetic effect in the careers warrants the survival of the variant under selection pressures. Functionally or metabolically related modifier variants make an epistatic network module and dozens of modules may be involved in the phenotype. To assess the significance of stochastic epistasis, a simplified module-based model was employed. The individual repertoire of the modifier variants in a module also participates in the genetic individuality which determines the genetic contribution of each modifier in the career. Because the entire contribution of a module to the phenotypic outcome is consequently unpredictable in the model, the module effect represents the total contribution of the related modifiers as a stochastic unit in the simulations. As a result, the intrinsic compatibility between distributional robustness and quantitative changeability could mathematically be simulated using the model. The artificial normal distribution shape in large-sized simulations was preserved in each generation even if the lowest fitness tail was un-reproductive. The robustness of normality beyond generations is analogous to the real situations of human complex diversity including neurodevelopmental conditions. The repeated regeneration of the un-reproductive extreme tail may be inevitable for the reproductive majority's competence to survive and change, suggesting implications of the extremes for others. Further model-simulations to

Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

DEFF Research Database (Denmark)

Haack-Sørensen, Mandana; Hansen, Susanne Kofoed; Hansen, Louise

2013-01-01

BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit...... differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which...... persists independent of cell density can be used for clinical therapies....

Phenotypically non-suppressive cells predominate among FoxP3-positive cells in oral lichen planus.

Science.gov (United States)

Schreurs, Olav; Karatsaidis, Andreas; Schenck, Karl

2016-11-01

Oral lichen planus (OLP) is a common T-cell-dominated oral chronic inflammatory disease occurring in periods of remission, quiescence, activity with pronounced inflammation, and acute ulceration. Cell infiltrates in OLP contain varying numbers of CD4 + T cells expressing the transcription factor FoxP3. FoxP3 + CD4 + T cells are, however, a heterogeneous cell population containing suppressive and non-suppressive cells, and their distribution in infiltrates from OLP is unknown. Biopsies were taken from normal oral mucosa (n = 8) and OLP lesions (n = 19), and a set of in situ methods for the determination of the functional phenotype of FoxP3 + CD4 + T cells was applied. Numbers of FoxP3 + CD4 + T cells were highest in the atrophic form of the disease, yet low in the ulcerative form. The main FoxP3 + CD4 + T-cell population observed was FoxP3 + CD45RA - CD25 + CD45RO + and CD15s - , a phenotype delineating a non-suppressive subset. Numbers of cells with an actively suppressing phenotype (FoxP3 + CD45RA - CD25 + CD45RO + and CD15s + ) were, however, about twice as high in reticular lesions as compared with the atrophic form. Many FoxP3 + CD4 + T cells expressed T-bet, the hallmark transcription factor for IFN-γ-producing T cells, indicating that they may enhance immune and inflammatory responses rather than suppress them. The absence of actively suppressing FoxP3 + CD4 + T cells may in part explain why OLP is a remarkably persisting condition, in spite of the presence of substantially high numbers of FoxP3 + CD4 + T cells. The findings emphasize that it is crucial to examine not only numbers but also functional phenotype of FoxP3 + CD4 + T cells in human tissues. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT)-Phenotypic Cells: Are They Cousins or Twins?

International Nuclear Information System (INIS)

Kong, Dejuan; Li, Yiwei; Wang, Zhiwei; Sarkar, Fazlul H.

2011-01-01

Cancer stem cells (CSCs) are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT), induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT-Phenotypic Cells: Are They Cousins or Twins?

Directory of Open Access Journals (Sweden)

Fazlul H. Sarkar

2011-02-01

Full Text Available Cancer stem cells (CSCs are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT, induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors.

The Phenotypic Fate of Bone Marrow-Derived Stem Cells in Acute Kidney Injury

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Guowei Feng

2013-11-01

Full Text Available Background: Despite increasing attention on the role of bone marrow derived stem cells in repair or rejuvenation of tissues and organs, cellular mechanisms of such cell-based therapy remain poorly understood. Methods: We reconstituted hematopoiesis in recipient C57BL/6J mice by transplanting syngeneic GFP+ bone marrow (BM cells. Subsequently, the recipients received subcutaneous injection of granulocyte-colony stimulating factor (G-CSF and were subjected to acute renal ischemic injury. Flow cytometry and immunostaining were performed at various time points to assess engraftment and phenotype of BM derived stem cells. Results: Administration of G-CSF increased the release of BM derived stem cells into circulation and enhanced the ensuing recruitment of BM derived stem cells into injured kidney. During the second month post injury, migrated BM derived stem cells lost hematopoietic phenotype (CD45 but maintained the expression of other markers (Sca-1, CD133 and CD44, suggesting their potential of transdifferentiation into renal stem cells. Moreover, G-CSF treatment enhanced the phenotypic conversion. Conclusion: Our work depicted a time-course dependent transition of phenotypic characteristics of BM derived stem cells, demonstrated the existence of BM derived stem cells in damaged kidney and revealed the effects of G-CSF on cell transdifferentiation.

Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERβ and glucocorticoid receptor [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Bianca De Leo

2017-05-01

Full Text Available Background: Human mast cells (MCs are long-lived tissue-resident immune cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1 To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2 To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ, progesterone (PR and glucocorticoids (GR. Methods: Tissue samples from women (n=46 were used for RNA extraction or fixed for immunohistochemistry. Results: Messenger RNAs encoded by TPSAB1 (tryptase and CMA1 (chymase were detected in endometrial tissue homogenates. Immunohistochemistry revealed the relative abundance of tryptase MCs was myometrium>basal endometrium>functional endometrium. We show for the first time that uterine MCs are predominantly of the classical MC subtypes: (positive, +; negative, - tryptase+/chymase- and tryptase+/chymase+, but a third subtype was also identified (tryptase-/chymase+. Tryptase+ MCs were of an ERβ+/ERα-/PR-/GR+ phenotype mirroring other uterine immune cell populations, including natural killer cells. Conclusions: Endometrial tissue resident immune MCs have three protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors

Delayed reproductive death as a dominant phenotype in cell clones surviving X-irradiation

International Nuclear Information System (INIS)

Chang, W.P.; Little, J.B.

1992-01-01

Residual damage manifested as reduced cloning efficiency was observed in many of the cloned progeny of Chinese hamster ovary (CHO) cells and human carcinoma SQ-20B cells surviving X-irradiation. This stable phenotype, which we have termed delayed reproductive death, persisted for >50 generations of cell replication post-irradiation. Clones showing this phenotype were aneuploid, and formed colonies with a high proportion of giant cells. By somatic cell hybridization of CHO clones, the delayed reproductive death phenotype was found to be a dominant trait; the cloning efficiency of hybrid clones was persistently depressed, as compared with that of control hybrid cells. These results suggest that delayed reproductive death represents a specific cellular response that may persist in some of the progeny of mammalian cells for long periods after X-irradiation. (author)

The emerging phenotype of the testicular carcinoma in situ germ cell

DEFF Research Database (Denmark)

Rajpert-De Meyts, Ewa; Bartkova, Jirina; Samson, Michel

2003-01-01

This review summarises the existing knowledge on the phenotype of the carcinoma in situ (CIS) cell. CIS is a common pre-invasive precursor of testicular germ cell tumours of adolescents and young adults. These tumours display a variety of histological forms. Classical seminoma proliferates along...... of differentiation and pluripotency, CIS cells found in adult patients seem to be predestined for further malignant progression into one or the other of the two main types of overt tumours. A new concept of phenotypic continuity of differentiation of germ cells along germinal lineage with a gradual loss of embryonic...

Transparent solar cell window module

Energy Technology Data Exchange (ETDEWEB)

Chau, Joseph Lik Hang; Chen, Ruei-Tang; Hwang, Gan-Lin; Tsai, Ping-Yuan [Nanopowder and Thin Film Technology Center, ITRI South, Industrial Technology Research Institute, Tainan County 709 (China); Lin, Chien-Chu [I-Lai Acrylic Corporation, Tainan City (China)

2010-03-15

A transparent solar cell window module based on the integration of traditional silicon solar cells and organic-inorganic nanocomposite material was designed and fabricated. The transparent solar cell window module was composed of a nanocomposite light-guide plate and traditional silicon solar cells. The preparation of the nanocomposite light-guide plate is easy without modification of the traditional casting process, the nanoparticles sol can be added directly to the polymethyl methacrylate (PMMA) monomer syrup during the process. The solar energy collected by this window can be used to power up small household electrical appliances. (author)

Oxygen Modulates Human Decidual Natural Killer Cell Surface Receptor Expression and Interactions with Trophoblasts1

Science.gov (United States)

Wallace, Alison E.; Goulwara, Sonu S.; Whitley, Guy S.; Cartwright, Judith E.

2014-01-01

Decidual natural killer (dNK) cells have been shown to both promote and inhibit trophoblast behavior important for decidual remodeling in pregnancy and have a distinct phenotype compared to peripheral blood NK cells. We investigated whether different levels of oxygen tension, mimicking the physiological conditions of the decidua in early pregnancy, altered cell surface receptor expression and activity of dNK cells and their interactions with trophoblast. dNK cells were isolated from terminated first-trimester pregnancies and cultured in oxygen tensions of 3%, 10%, and 21% for 24 h. Cell surface receptor expression was examined by flow cytometry, and the effects of secreted factors in conditioned medium (CM) on the trophoblast cell line SGHPL-4 were assessed in vitro. SGHPL-4 cells treated with dNK cell CM incubated in oxygen tensions of 10% were significantly more invasive (P cells treated with dNK cell CM incubated in oxygen tensions of 3% or 21%. After 24 h, a lower percentage of dNK cells expressed CD56 at 21% oxygen (P cells expressed NKG2D at 10% oxygen (P oxygen tensions, with large patient variation. This study demonstrates dNK cell phenotype and secreted factors are modulated by oxygen tension, which induces changes in trophoblast invasion and endovascular-like differentiation. Alterations in dNK cell surface receptor expression and secreted factors at different oxygen tensions may represent regulation of function within the decidua during the first trimester of pregnancy. PMID:25232021

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

Science.gov (United States)

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

International Nuclear Information System (INIS)

Spangrude, G.J.; Scollay, R.

1990-01-01

To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes

Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation

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Paula A. Baldión

2018-01-01

Full Text Available Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-β1, and OLC expanded after trypsinization (EXP-21 were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry.

Nicotine can skew the characterization of the macrophage type-1 (MΦ1) phenotype differentiated with granulocyte-macrophage colony-stimulating factor to the MΦ2 phenotype

International Nuclear Information System (INIS)

Yanagita, Manabu; Kobayashi, Ryohei; Murakami, Shinya

2009-01-01

Macrophages (MΦs) exhibit functional heterogeneity and plasticity in the local microenvironment. Recently, it was reported that MΦs can be divided into proinflammatory MΦs (MΦ1) and anti-inflammatory MΦs (MΦ2) based on their polarized functional properties. Here, we report that nicotine, the major ingredient of cigarette smoke, can modulate the characteristics of MΦ1. Granulocyte-macrophage colony-stimulating factor-driven MΦ1 with nicotine (Ni-MΦ1) showed the phenotypic characteristics of MΦ2. Like MΦ2, Ni-MΦ1 exhibited antigen-uptake activities. Ni-MΦ1 suppressed IL-12, but maintained IL-10 and produced high amounts of MCP-1 upon lipopolysaccharide stimulation compared with MΦ1. Moreover, we observed strong proliferative responses of T cells to lipopolysaccharide-stimulated MΦ1, whereas Ni-MΦ1 reduced T cell proliferation and inhibited IFN-γ production by T cells. These results suggest that nicotine can change the functional characteristics of MΦ and skew the MΦ1 phenotype to MΦ2. We propose that nicotine is a potent regulator that modulates immune responses in microenvironments.

Ex vivo modulation of the Foxo1 phosphorylation state does not lead to dysfunction of T regulatory cells.

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Kristen Kelley Penberthy

Full Text Available Peripheral regulatory CD4+ T cells (Treg cells prevent maladaptive inflammatory responses to innocuous foreign antigens. Treg cell dysfunction has been linked to many inflammatory diseases, including allergic airway inflammation. Glucocorticoids that are used to treat allergic airway inflammation and asthma are thought to work in part by promoting Treg cell differentiation; patients who are refractory to these drugs have defective induction of anti-inflammatory Treg cells. Previous observations suggest that Treg cells deficient in the transcription factor FoxO1 are pro-inflammatory, and that FoxO1 activity is regulated by its phosphorylation status and nuclear localization. Here, we asked whether altering the phosphorylation state of FoxO1 through modulation of a regulatory phosphatase might affect Treg cell function. In a mouse model of house dust mite-induced allergic airway inflammation, we observed robust recruitment of Treg cells to the lungs and lymph nodes of diseased mice, without an apparent increase in the Treg cytokine interleukin-10 in the airways. Intriguingly, expression of PP2A, a serine/threonine phosphatase linked to the regulation of FoxO1 phosphorylation, was decreased in the mediastinal lymph nodes of HDM-treated mice, mirroring the decreased PP2A expression seen in peripheral blood monocytes of glucocorticoid-resistant asthmatic patients. When we asked whether modulation of PP2A activity alters Treg cell function via treatment with the PP2A inhibitor okadaic acid, we observed increased phosphorylation of FoxO1 and decreased nuclear localization. However, dysregulation of FoxO1 did not impair Treg cell differentiation ex vivo or cause Treg cells to adopt a pro-inflammatory phenotype. Moreover, inhibition of PP2A activity did not affect the suppressive function of Treg cells ex vivo. Collectively, these data suggest that modulation of the phosphorylation state of FoxO1 via PP2A inhibition does not modify Treg cell function ex

GenomeRNAi: a database for cell-based RNAi phenotypes.

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Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

International Nuclear Information System (INIS)

Yamagishi, Naoko; Teshima-Kondo, Shigetada; Masuda, Kiyoshi; Nishida, Kensei; Kuwano, Yuki; Dang, Duyen T; Dang, Long H; Nikawa, Takeshi; Rokutan, Kazuhito

2013-01-01

Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF

Association of Immunological Cell Profiles with Specific Clinical Phenotypes of Scleroderma Disease

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Calzada, David; Mayayo, Teodoro; González-Rodríguez, María Luisa; Rabasco, Antonio María; Lahoz, Carlos

2014-01-01

This study aimed to search the correlation among immunological profiles and clinical phenotypes of scleroderma in well-characterized groups of scleroderma patients, comparing forty-nine scleroderma patients stratified according to specific clinical phenotypes with forty-nine healthy controls. Five immunological cell subpopulations (B, CD4+ and CD8+ T-cells, NK, and monocytes) and their respective stages of apoptosis and activation were analyzed by flow cytometry, in samples of peripheral blood mononuclear cells (PBMCs). Analyses of results were stratified according to disease stage, time since the diagnosis, and visceral damage (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time of treatment with corticosteroids. An increase in the percentages of monocytes and a decrease in the B cells were mainly related to the disease progression. A general apoptosis decrease was found in all phenotypes studied, except in localized scleroderma. An increase of B and NK cells activation was found in patients diagnosed more than 10 years ago. Specific cell populations like monocytes, NK, and B cells were associated with the type of affected organ. This study shows how, in a heterogeneous disease, proper patient's stratification according to clinical phenotypes allows finding specific cellular profiles. Our data may lead to improvements in the knowledge of prognosis factors and to aid in the analysis of future specific therapies. PMID:24818126

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

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Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

2016-01-01

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

Contribution of neural cell death to depressive phenotypes of streptozotocin-induced diabetic mice

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Cheng Chen

2014-06-01

Full Text Available Major depression disorder (MDD or depression is highly prevalent in individuals with diabetes, and the depressive symptoms are more severe and less responsive to antidepressant therapies in these patients. The underlying mechanism is little understood. We hypothesized that the pathophysiology of comorbid depression was more complex than that proposed for MDD and that neural cell death played a role in the disease severity. To test this hypothesis, we generated streptozotocin (STZ-induced diabetic mice. These mice had blood glucose levels threefold above controls and exhibited depressive phenotypes as judged by a battery of behavioral tests, thus confirming the comorbidity in mice. Immunohistological studies showed markedly increased TUNEL-positive cells in the frontal cortex and hippocampus of the comorbid mice, indicating apoptosis. This finding was supported by increased caspase-3 and decreased Bcl-2 proteins in these brain regions. In addition, the serum brain-derived neurotrophic factor (BDNF level of comorbid mice was reduced compared with controls, further supporting the neurodegenerative change. Mechanistic analyses showed an increased expression of mitochondrial fission genes fission protein 1 (Fis1 and dynamin-related protein 1 (Drp1, and a decreased expression of mitochondrial fusion genes mitofusin 1 (Mfn1, mitofusin 2 (Mfn2 and optical atrophy 1 (Opa1. Representative assessment of the proteins Drp1 and Mfn2 mirrored the mRNA changes. The data demonstrated that neural cell death was associated with the depressive phenotype of comorbid mice and that a fission-dominant expression of genes and proteins mediating mitochondrial dynamics played a role in the hyperglycemia-induced cell death. The study provides new insight into the disease mechanism and could aid the development of novel therapeutics aimed at providing neuroprotection by modulating mitochondrial dynamics to treat comorbid depression with diabetes.

T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

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Kostas Patas

2018-02-01

Full Text Available While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR repertoire in MDD. For this cross-sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20, who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20. T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.

High-Dimensional Phenotyping Identifies Age-Emergent Cells in Human Mammary Epithelia

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Fanny A. Pelissier Vatter

2018-04-01

Full Text Available Summary: Aging is associated with tissue-level changes in cellular composition that are correlated with increased susceptibility to disease. Aging human mammary tissue shows skewed progenitor cell potency, resulting in diminished tumor-suppressive cell types and the accumulation of defective epithelial progenitors. Quantitative characterization of these age-emergent human cell subpopulations is lacking, impeding our understanding of the relationship between age and cancer susceptibility. We conducted single-cell resolution proteomic phenotyping of healthy breast epithelia from 57 women, aged 16–91 years, using mass cytometry. Remarkable heterogeneity was quantified within the two mammary epithelial lineages. Population partitioning identified a subset of aberrant basal-like luminal cells that accumulate with age and originate from age-altered progenitors. Quantification of age-emergent phenotypes enabled robust classification of breast tissues by age in healthy women. This high-resolution mapping highlighted specific epithelial subpopulations that change with age in a manner consistent with increased susceptibility to breast cancer. : Vatter et al. find that single-cell mass cytometry of human mammary epithelial cells from 57 women, from 16 to 91 years old, depicts an in-depth phenotyping of aging mammary epithelia. Subpopulations of altered luminal and progenitor cells that accumulate with age may be at increased risk for oncogenic transformation. Keywords: human mammary epithelia, aging, mass cytometry, single-cell analysis, heterogeneity, breast cancer

Phenotype heterogeneity in cancer cell populations

International Nuclear Information System (INIS)

Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean; Escargueil, Alexandre; Lorenzi, Tommaso; Lorz, Alexander; Trélat, Emmanuel

2016-01-01

Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as “bet hedging” of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

Phenotype heterogeneity in cancer cell populations

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Almeida, Luis [CNRS UMR 7598, LJLL, & INRIA MAMBA team, Sorbonne Universités, UPMC Univ Paris 06, Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, luis@ann.jussieu.fr (France); Chisholm, Rebecca [School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia, rebecca.chisholm@gmail.com (Australia); Clairambault, Jean [INRIA MAMBA team & LJLL, UMR 7598, Sorbonne Universités, UPMC Univ Paris 06, Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, jean.clairambault@inria.fr, Corresponding author (France); Escargueil, Alexandre [INSERM “Cancer Biology and Therapeutics”, Sorbonne Universités, UPMC Univ Paris 06, UMR-S 938, CDR St Antoine, Hôpital St Antoine, 184 Fbg. St Antoine, 75571 Paris cedex 12, France, alexandre.escargueil@upmc.fr (France); Lorenzi, Tommaso [CMLA, ENS Cachan, 61, Av. du Président Wilson, 94230 Cachan cedex & INRIA MAMBA team, & LJLL, UMR 7598, UPMC Univ Paris 06, Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, tommaso.lorenzi@gmail.com (France); Lorz, Alexander [Sorbonne Universités, UPMC Univ Paris 06, LJLL, UMR 7598 & INRIA Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, alex.lorz@ann.jussieu.fr (France); Trélat, Emmanuel [Institut Universitaire de France, Sorbonne Universités, UPMC Univ Paris 06, LJLL, UMR 7598, Boîte courrier 187, UPMC Univ Paris 06, 4 Pl. Jussieu, 75252 Paris cedex 05, France, emmanuel.trelat@upmc.fr (France)

2016-06-08

Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as “bet hedging” of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

Phenotype heterogeneity in cancer cell populations

Science.gov (United States)

Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean; Escargueil, Alexandre; Lorenzi, Tommaso; Lorz, Alexander; Trélat, Emmanuel

2016-06-01

Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as "bet hedging" of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

A Quantitative Analysis of Photovoltaic Modules Using Halved Cells

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S. Guo

2013-01-01

Full Text Available In a silicon wafer-based photovoltaic (PV module, significant power is lost due to current transport through the ribbons interconnecting neighbour cells. Using halved cells in PV modules is an effective method to reduce the resistive power loss which has already been applied by some major PV manufacturers (Mitsubishi, BP Solar in their commercial available PV modules. As a consequence, quantitative analysis of PV modules using halved cells is needed. In this paper we investigate theoretically and experimentally the difference between modules made with halved and full-size solar cells. Theoretically, we find an improvement in fill factor of 1.8% absolute and output power of 90 mW for the halved cell minimodule. Experimentally, we find an improvement in fill factor of 1.3% absolute and output power of 60 mW for the halved cell module. Also, we investigate theoretically how this effect confers to the case of large-size modules. It is found that the performance increment of halved cell PV modules is even higher for high-efficiency solar cells. After that, the resistive loss of large-size modules with different interconnection schemes is analysed. Finally, factors influencing the performance and cost of industrial halved cell PV modules are discussed.

Cell and small animal models for phenotypic drug discovery

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Szabo M

2017-06-01

Full Text Available Mihaly Szabo,1 Sara Svensson Akusjärvi,1 Ankur Saxena,1 Jianping Liu,2 Gayathri Chandrasekar,1 Satish S Kitambi1 1Department of Microbiology Tumor, and Cell Biology, 2Department of Biochemistry and Biophysics, Karolinska Institutet, Solna, Sweden Abstract: The phenotype-based drug discovery (PDD approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery. Keywords: phenotype, screening, PDD, discovery, zebrafish, drug

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

Science.gov (United States)

Floren, Michael; Bonani, Walter; Dharmarajan, Anirudh; Motta, Antonella; Migliaresi, Claudio; Tan, Wei

2016-02-01

demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

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Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Stem Cell Differentiation Stage Factors and Their Role in Triggering Symmetry Breaking Processes during Cancer Development: A Quantum Field Theory Model for Reprogramming Cancer Cells to Healthy Phenotypes.

Science.gov (United States)

Biava, Pier Mario; Burigana, Fabio; Germano, Roberto; Kurian, Philip; Verzegnassi, Claudio; Vitiello, Giuseppe

2017-09-20

A long history of research has pursued the use of embryonic factors isolated during cell differentiation processes for the express purpose of transforming cancer cells back to healthy phenotypes. Recent results have clarified that the substances present at different stages of cell differentiation-which we call stem cell differentiation stage factors (SCDSFs)-are proteins with low molecular weight and nucleic acids that regulate genomic expression. The present review summarizes how these substances, taken at different stages of cellular maturation, are able to retard proliferation of many human tumor cell lines and thereby reprogram cancer cells to healthy phenotypes. The model presented here is a quantum field theory (QFT) model in which SCDSFs are able to trigger symmetry breaking processes during cancer development. These symmetry breaking processes, which lie at the root of many phenomena in elementary particle physics and condensed matter physics, govern the phase transitions of totipotent cells to higher degrees of diversity and order, resulting in cell differentiation. In cancers, which share many genomic and metabolic similarities with embryonic stem cells, stimulated re-differentiation often signifies the phenotypic reversion back to health and non-proliferation. In addition to acting on key components of the cellular cycle, SCDSFs are able to reprogram cancer cells by delicately influencing the cancer microenvironment, modulating the electrochemistry and thus the collective electrodynamic behaviors between dipole networks in biomacromolecules and the interstitial water field. Coherent effects in biological water, which are derived from a dissipative QFT framework, may offer new diagnostic and therapeutic targets at a systemic level, before tumor instantiation occurs in specific tissues or organs. Thus, by including the environment as an essential component of our model, we may push the prevailing paradigm of mutation-driven oncogenesis toward a closer

3D collagen fibrillar microstructure guides pancreatic cancer cell phenotype and serves as a critical design parameter for phenotypic models of EMT.

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T J Puls

Full Text Available Pancreatic cancer, one of the deadliest cancers, is characterized by high rates of metastasis and intense desmoplasia, both of which are associated with changes in fibrillar type I collagen composition and microstructure. Epithelial to mesenchymal transition (EMT, a critical step of metastasis, also involves a change in extracellular matrix (ECM context as cells detach from basement membrane (BM and engage interstitial matrix (IM. The objective of this work was to develop and apply an in-vitro three-dimensional (3D tumor-ECM model to define how ECM composition and biophysical properties modulate pancreatic cancer EMT. Three established pancreatic ductal adenocarcinoma (PDAC lines were embedded within 3D matrices prepared with type I collagen Oligomer (IM at various fibril densities to control matrix stiffness or Oligomer and Matrigel combined at various ratios while maintaining constant matrix stiffness. Evaluation of cell morphology and protein expression at both the cellular- and population-levels revealed a spectrum of matrix-driven EMT phenotypes that were dependent on ECM composition and architecture as well as initial PDAC phenotype. In general, exposure to fibrillar IM was sufficient to drive EMT, with cells displaying spindle-shaped morphology and mesenchymal markers, and non-fibrillar BM promoted more epithelial behavior. When cultured within low density Oligomer, only a subpopulation of epithelial BxPC-3 cells displayed EMT while mesenchymal MiaPaCa-2 cells displayed more uniform spindle-shaped morphologies and mesenchymal marker expression. Interestingly, as IM fibril density increased, associated changes in spatial constraints and matrix stiffness resulted in all PDAC lines growing as tight clusters; however mesenchymal marker expression was maintained. Collectively, the comparison of these results to other in-vitro tumor models highlights the role of IM fibril microstructure in guiding EMT heterogeneity and showcases the potential

Reprogramming of cell junction modules during stepwise epithelial to mesenchymal transition and accumulation of malignant features in vitro in a prostate cell model

International Nuclear Information System (INIS)

Ke, Xi-song; Li, Wen-cheng; Hovland, Randi; Qu, Yi; Liu, Run-hui; McCormack, Emmet; Thorsen, Frits; Olsen, Jan Roger; Molven, Anders; Kogan-Sakin, Ira; Rotter, Varda; Akslen, Lars A.; Oyan, Anne Margrete; Kalland, Karl-Henning

2011-01-01

Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.

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Augustsson, Per; Karlsen, Jonas T; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

2016-05-16

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.

Excess 25-hydroxyvitamin D3 exacerbates tubulointerstitial injury in mice by modulating macrophage phenotype.

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Kusunoki, Yasuo; Matsui, Isao; Hamano, Takayuki; Shimomura, Akihiro; Mori, Daisuke; Yonemoto, Sayoko; Takabatake, Yoshitsugu; Tsubakihara, Yoshiharu; St-Arnaud, René; Isaka, Yoshitaka; Rakugi, Hiromi

2015-11-01

Vitamin D hydroxylated at carbon 25 (25(OH)D) is generally recognized as a precursor of active vitamin D. Despite its low affinity for the vitamin D receptor (VDR), both deficient and excessive 25(OH)D levels are associated with poor clinical outcomes. Here we studied direct effects of 25(OH)D3 on the kidney using 25(OH)D-1α-hydroxylase (CYP27B1) knockout mice. The effects of 25(OH)D3 on unilateral ureteral obstruction were analyzed as proximal tubular cells and macrophages are two major cell types that take up 25(OH)D and contribute to the pathogenesis of kidney injury. Excess 25(OH)D3 in obstructed mice worsened oxidative stress and tubulointerstitial fibrosis, whereas moderate levels of 25(OH)D3 had no effects. The exacerbating effects of excess 25(OH)D3 were abolished in CYP27B1/VDR double-knockout mice and in macrophage-depleted CYP27B1 knockout mice. Excess 25(OH)D3 upregulated both M1 marker (TNF-α) and M2 marker (TGF-β1) levels of kidney-infiltrating macrophages. In vitro analyses verified that excess 25(OH)D3 directly upregulated TNF-α and TGF-β1 in cultured macrophages but not in tubular cells. TNF-α and 25(OH)D3 cooperatively induced oxidative stress by upregulating iNOS in tubular cells. Aggravated tubulointerstitial fibrosis in mice with excess 25(OH)D3 indicated that macrophage-derived TGF-β1 also had a key role in the pathogenesis of surplus 25(OH)D3. Thus, excess 25(OH)D3 worsens tubulointerstitial injury by modulating macrophage phenotype.

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells

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Johansson, Elinn; Grassi, Elisa S.; Pantazopoulou, Vasiliki

2017-01-01

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell...... marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature...... correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic...

Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling

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Ilenia De Luca

2016-09-01

Full Text Available Understanding the mechanisms by which mesenchymal stromal cells (MSCs interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3 with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.

Modulation of Hematopoietic Lineage Specification Impacts TREM2 Expression in Microglia-Like Cells Derived From Human Stem Cells.

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Amos, Peter J; Fung, Susan; Case, Amanda; Kifelew, Jerusalem; Osnis, Leah; Smith, Carole L; Green, Kevin; Naydenov, Alipi; Aloi, Macarena; Hubbard, Jesse J; Ramakrishnan, Aravind; Garden, Gwenn A; Jayadev, Suman

2017-01-01

Microglia are the primary innate immune cell type in the brain, and their dysfunction has been linked to a variety of central nervous system disorders. Human microglia are extraordinarily difficult to obtain for experimental investigation, limiting our ability to study the impact of human genetic variants on microglia functions. Previous studies have reported that microglia-like cells can be derived from human monocytes or pluripotent stem cells. Here, we describe a reproducible relatively simple method for generating microglia-like cells by first deriving embryoid body mesoderm followed by exposure to microglia relevant cytokines. Our approach is based on recent studies demonstrating that microglia originate from primitive yolk sac mesoderm distinct from peripheral macrophages that arise during definitive hematopoiesis. We hypothesized that functional microglia could be derived from human stem cells by employing BMP-4 mesodermal specification followed by exposure to microglia-relevant cytokines, M-CSF, GM-CSF, IL-34, and TGF-β. Using immunofluorescence microscopy, flow cytometry, and reverse transcription polymerase chain reaction, we observed cells with microglia morphology expressing a repertoire of markers associated with microglia: Iba1, CX3CR1, CD11b, TREM2, HexB, and P2RY12. These microglia-like cells maintain myeloid functional phenotypes including Aβ peptide phagocytosis and induction of pro-inflammatory gene expression in response to lipopolysaccharide stimulation. Addition of small molecules BIO and SB431542, previously demonstrated to drive definitive hematopoiesis, resulted in decreased surface expression of TREM2. Together, these data suggest that mesodermal lineage specification followed by cytokine exposure produces microglia-like cells in vitro from human pluripotent stem cells and that this phenotype can be modulated by factors influencing hematopoietic lineage in vitro.

Integrated modulation of phorbol ester-induced Raf activation in EL4 lymphoma cells.

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Han, Shujie; Meier, Kathryn E

2009-05-01

The EL4 murine lymphoma cell line exists in variant phenotypes that differ with respect to responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA1). Previous work showed that "PMA-sensitive" cells, characterized by a high magnitude of PMA-induced Erk activation, express RasGRP, a phorbol ester receptor that directly activates Ras. In "PMA-resistant" and "intermediate" EL4 cell lines, PMA induces Erk activation to lesser extents, but with a greater response in intermediate cells. In the current study, these cell lines were used to examine mechanisms of Raf-1 modulation. Phospho-specific antibodies were utilized to define patterns and kinetics of Raf-1 phosphorylation on several sites. Further studies showed that Akt is constitutively activated to a greater extent in PMA-resistant than in PMA-sensitive cells, and also to a greater extent in resistant than intermediate cells. Akt negatively regulates Raf-1 activation (Ser259), partially explaining the difference between resistant and intermediate cells. Erk activation exerts negative feedback on Raf-1 (Ser289/296/301), thus resulting in earlier termination of the signal in cells with a higher level of Erk activation. RKIP, a Raf inhibitory protein, is expressed at higher levels in resistant cells than in sensitive or intermediate cells. Knockdown of RKIP increases Erk activation and also negative feedback. In conclusion, this study delineates Raf-1 phosphorylation events occurring in response to PMA in cell lines with different extents of Erk activation. Variations in the levels of expression and activation of multiple signaling proteins work in an integrated fashion to modulate the extent and duration of Erk activation.

Radiation-induced senescence-like phenotype in proliferating and plateau-phase vascular endothelial cells

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Igarashi, Kaori; Sakimoto, Ippei; Kataoka, Keiko; Ohta, Keisuke; Miura, Masahiko

2007-01-01

The effects of ionizing radiation (IR) on tumor angiogenesis still remain largely unknown. In this study, we found that IR (8 Gy) induces a high-frequency (80-90%) senescence-like phenotype in vascular endothelial cells (ECs) undergoing exponential growth. This finding allowed us to characterize the IR-induced senescence-like (IRSL) phenotype by examining the gene expression profiles and in vitro angiogenic activities of these ECs. The expression levels of genes associated with cell cycle progression and DNA replication were remarkably reduced in the IRSL ECs. Additionally, the in vitro invasion and migration activities of these cells through Matrigel were significantly suppressed. We also found that confluent ECs exhibited a high-frequency IRSL phenotype when they were replated immediately after irradiation, whereas incubation in plateau-phase conditions reduced the induction of this phenotype and enhanced colony formation. The kinetics of DNA double-strand break repair, which showed a faster time course in confluent ECs than in growing ECs, may contribute to the protective mechanism associated with the IRSL phenotype. These results imply that the IRSL phenotype may be important for determining the angiogenic activity of ECs following irradiation. The present study should contribute to the understanding of the effects of IR on tumor angiogenesis

In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

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Timraz, Sara B.H., E-mail: sara.timraz@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Farhat, Ilyas A.H., E-mail: ilyas.farhat@outlook.com [Department of Applied Mathematics and Sciences, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Alhussein, Ghada, E-mail: ghada.alhussein@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Christoforou, Nicolas, E-mail: nicolas.christoforou@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Department of Biomedical Engineering, Duke University, Durham, NC 27708 (United States); Teo, Jeremy C.M., E-mail: jeremy.teo@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates)

2016-05-01

In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, and reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.

In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

International Nuclear Information System (INIS)

Timraz, Sara B.H.; Farhat, Ilyas A.H.; Alhussein, Ghada; Christoforou, Nicolas; Teo, Jeremy C.M.

2016-01-01

In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, and reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.

Red blood cell phenotype prevalence in blood donors who self-identify as Hispanic

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Sheppard, Chelsea A; Bolen, Nicole L; Eades, Beth

2017-01-01

CONCLUSIONS: Molecular genotyping platforms provide a quick, high-throughput method for identifying red blood cell units for patients on extended phenotype-matching protocols, such as those with sickle cell disease or thalassemia. Most of the antigen prevalence data reported are for non-Hispanic ......CONCLUSIONS: Molecular genotyping platforms provide a quick, high-throughput method for identifying red blood cell units for patients on extended phenotype-matching protocols, such as those with sickle cell disease or thalassemia. Most of the antigen prevalence data reported are for non...

Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma

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Adrian Biddle

2016-02-01

Full Text Available Cancer stem cells (CSCs drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT and mesenchymal-to-epithelial transition (MET to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC, we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44highEpCAMlow/−CD24+ cell surface marker profile. Treatment with TGFβ and retinoic acid (RA enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma.

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Biddle, Adrian; Gammon, Luke; Liang, Xiao; Costea, Daniela Elena; Mackenzie, Ian C

2016-02-01

Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44(high)EpCAM(low/-) CD24(+) cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

A limited role for p53 in modulating the immediate phenotype of Apc loss in the intestine

International Nuclear Information System (INIS)

Reed, Karen R; Meniel, Valerie S; Marsh, Victoria; Cole, Alicia; Sansom, Owen J; Clarke, Alan R

2008-01-01

p53 is an important tumour suppressor with a known role in the later stages of colorectal cancer, but its relevance to the early stages of neoplastic initiation remains somewhat unclear. Although p53-dependent regulation of Wnt signalling activity is known to occur, the importance of these regulatory mechanisms during the early stages of intestinal neoplasia has not been demonstrated. We have conditionally deleted the Adenomatous Polyposis coli gene (Apc) from the adult murine intestine in wild type and p53 deficient environments and subsequently compared the phenotype and transcriptome profiles in both genotypes. Expression of p53 was shown to be elevated following the conditional deletion of Apc in the adult small intestine. Furthermore, p53 status was shown to impact on the transcription profile observed following Apc loss. A number of key Wnt pathway components and targets were altered in the p53 deficient environment. However, the aberrant phenotype observed following loss of Apc (rapid nuclear localisation of β-catenin, increased levels of DNA damage, nuclear atypia, perturbed cell death, proliferation, differentiation and migration) was not significantly altered by the absence of p53. p53 related feedback mechanisms regulating Wnt signalling activity are present in the intestine, and become activated following loss of Apc. However, the physiological Wnt pathway regulation by p53 appears to be overwhelmed by Apc loss and consequently the activity of these regulatory mechanisms is not sufficient to modulate the immediate phenotypes seen following Apc loss. Thus we are able to provide an explanation to the apparent contradiction that, despite having a Wnt regulatory capacity, p53 loss is not associated with early lesion development

Laminated photovoltaic modules using back-contact solar cells

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Gee, James M.; Garrett, Stephen E.; Morgan, William P.; Worobey, Walter

1999-09-14

Photovoltaic modules which comprise back-contact solar cells, such as back-contact crystalline silicon solar cells, positioned atop electrically conductive circuit elements affixed to a planar support so that a circuit capable of generating electric power is created. The modules are encapsulated using encapsulant materials such as EVA which are commonly used in photovoltaic module manufacture. The module designs allow multiple cells to be electrically connected in a single encapsulation step rather than by sequential soldering which characterizes the currently used commercial practices.

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

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Omlor, G W; Nerlich, A G; Tirlapur, U K; Urban, J P; Guehring, T

2014-12-01

Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. 3D

CaV1.3 L-type Ca2+ channels modulate depression-like behaviour in mice independent of deaf phenotype.

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Busquet, Perrine; Nguyen, Ngoc Khoi; Schmid, Eduard; Tanimoto, Naoyuki; Seeliger, Mathias W; Ben-Yosef, Tamar; Mizuno, Fengxia; Akopian, Abram; Striessnig, Jörg; Singewald, Nicolas

2010-05-01

Mounting evidence suggests that voltage-gated L-type Ca2+ channels can modulate affective behaviour. We therefore explored the role of CaV1.3 L-type Ca2+ channels in depression- and anxiety-like behaviours using CaV1.3-deficient mice (CaV1.3-/-). We showed that CaV1.3-/- mice displayed less immobility in the forced swim test as well as in the tail suspension test, indicating an antidepressant-like phenotype. Locomotor activity in the home cage or a novel open-field test was not influenced. In the elevated plus maze (EPM), CaV1.3-/- mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however, not supported in the stress-induced hyperthermia test. By performing parallel experiments in Claudin 14 knockout mice (Cldn14-/-), which like CaV1.3-/- mice are congenitally deaf, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behaviour indicative of an anxiolytic phenotype was also found in the Cldn14-/- animals. Using electroretinography and visual behavioural tasks we demonstrated that at least in mice, CaV1.3 channels do not significantly contribute to visual function. However, marked morphological changes were revealed in synaptic ribbons in the outer plexiform layer of CaV1.3-/- retinas by immunohistochemistry suggesting a possible role of this channel type in structural plasticity at the ribbon synapse. Taken together, our findings indicate that CaV1.3 L-type Ca2+ channels modulate depression-like behaviour but are not essential for visual function. The findings raise the possibility that selective modulation of CaV1.3 channels could be a promising new therapeutic concept for the treatment of mood disorders.

RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

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De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini

2017-01-10

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by

Progastrin represses the alternative activation of human macrophages and modulates their influence on colon cancer epithelial cells.

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Carlos Hernández

Full Text Available Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive to M2 (tolerogenic, pro-tumoral. Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10 with normal amounts of M1-markers (CD86, IL12. Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization.

Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping.

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Juul, Troels; Palm, Fredrik; Nielsen, Per Mose; Bertelsen, Lotte Bonde; Laustsen, Christoffer

2017-08-01

It has been demonstrated that hyperpolarized 13 C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13 C MR is suitable for cells isolated from kidney tissue, without prior cell culture. Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13 C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. Ex vivo metabolic phenotyping using hyperpolarized 13 C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457-461, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells.

Directory of Open Access Journals (Sweden)

Cristiano Farace

Full Text Available The presence of cancer stem cells (CSCs or tumor-initiating cells can lead to cancer recurrence in a permissive cell-microenvironment interplay, promoting invasion in glioblastoma (GBM and neuroblastoma (NB. Extracellular matrix (ECM small leucine-rich proteoglycans (SLRPs play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs, SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN and lumican (LUM are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+ CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge

Microenvironmental Modulation of Decorin and Lumican in Temozolomide-Resistant Glioblastoma and Neuroblastoma Cancer Stem-Like Cells.

Science.gov (United States)

Farace, Cristiano; Oliver, Jaime Antonio; Melguizo, Consolacion; Alvarez, Pablo; Bandiera, Pasquale; Rama, Ana Rosa; Malaguarnera, Giulia; Ortiz, Raul; Madeddu, Roberto; Prados, Jose

2015-01-01

The presence of cancer stem cells (CSCs) or tumor-initiating cells can lead to cancer recurrence in a permissive cell-microenvironment interplay, promoting invasion in glioblastoma (GBM) and neuroblastoma (NB). Extracellular matrix (ECM) small leucine-rich proteoglycans (SLRPs) play multiple roles in tissue homeostasis by remodeling the extracellular matrix (ECM) components and modulating intracellular signaling pathways. Due to their pan-inhibitory properties against receptor tyrosine kinases (RTKs), SLRPs are reported to exert anticancer effects in vitro and in vivo. However, their roles seem to be tissue-specific and they are also involved in cancer cell migration and drug resistance, paving the way to complex different scenarios. The aim of this study was to determine whether the SLRPs decorin (DCN) and lumican (LUM) are recruited in cell plasticity and microenvironmental adaptation of differentiated cancer cells induced towards stem-like phenotype. Floating neurospheres were generated by applying CSC enrichment medium (neural stem cell serum-free medium, NSC SFM) to the established SF-268 and SK-N-SH cancer cell lines, cellular models of GBM and NB, respectively. In both models, the time-dependent synergistic activation of DCN and LUM was observed. The highest DCN and LUM mRNA/protein expression was detected after cell exposure to NSC SFM for 8/12 days, considering these cells as SLRP-expressing (SLRP+) CSC-like. Ultrastructural imaging showed the cellular heterogeneity of both the GBM and NB neurospheres and identified the inner living cells. Parental cell lines of both GBM and NB grew only in soft agar + NSC SFM, whereas the secondary neurospheres (originated from SLRP+ t8 CSC-like) showed lower proliferation rates than primary neurospheres. Interestingly, the SLRP+ CSC-like from the GBM and NB neurospheres were resistant to temozolomide (TMZ) at concentrations >750 μM. Our results suggest that GBM and NB CSC-like promote the activation of huge quantities

Immune modules shared by innate lymphoid cells and T cells.

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Robinette, Michelle L; Colonna, Marco

2016-11-01

In recent years, innate lymphoid cells (ILCs) have emerged as innate correlates to T cells. The similarities between ILCs and T cells indicate that lymphocytes of fundamentally distinct lineages can share core "immune modules" that encompass transcriptional circuitry and effector functions while using nonredundant complementary mechanisms of pattern recognition to enact these functions. We review modules currently recognized to be shared between ILCs and T cells. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The phenotypic equilibrium of cancer cells: From average-level stability to path-wise convergence.

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Niu, Yuanling; Wang, Yue; Zhou, Da

2015-12-07

The phenotypic equilibrium, i.e. heterogeneous population of cancer cells tending to a fixed equilibrium of phenotypic proportions, has received much attention in cancer biology very recently. In the previous literature, some theoretical models were used to predict the experimental phenomena of the phenotypic equilibrium, which were often explained by different concepts of stabilities of the models. Here we present a stochastic multi-phenotype branching model by integrating conventional cellular hierarchy with phenotypic plasticity mechanisms of cancer cells. Based on our model, it is shown that: (i) our model can serve as a framework to unify the previous models for the phenotypic equilibrium, and then harmonizes the different kinds of average-level stabilities proposed in these models; and (ii) path-wise convergence of our model provides a deeper understanding to the phenotypic equilibrium from stochastic point of view. That is, the emergence of the phenotypic equilibrium is rooted in the stochastic nature of (almost) every sample path, the average-level stability just follows from it by averaging stochastic samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Heat Shock Protein 47: A Novel Biomarker of Phenotypically Altered Collagen-Producing Cells

International Nuclear Information System (INIS)

Taguchi, Takashi; Nazneen, Arifa; Al-Shihri, Abdulmonem A.; Turkistani, Khadijah A.; Razzaque, Mohammed S.

2011-01-01

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that helps the molecular maturation of various types of collagens. A close association between increased expression of HSP47 and the excessive accumulation of collagens is found in various human and experimental fibrotic diseases. Increased levels of HSP47 in fibrotic diseases are thought to assist in the increased assembly of procollagen, and thereby contribute to the excessive deposition of collagens in fibrotic areas. Currently, there is not a good universal histological marker to identify collagen-producing cells. Identifying phenotypically altered collagen-producing cells is essential for the development of cell-based therapies to reduce the progression of fibrotic diseases. Since HSP47 has a single substrate, which is collagen, the HSP47 cellular expression provides a novel universal biomarker to identify phenotypically altered collagen-producing cells during wound healing and fibrosis. In this brief article, we explained why HSP47 could be used as a universal marker for identifying phenotypically altered collagen-producing cells

Vascular smooth muscle cell differentiation to an osteogenic phenotype involves matrix metalloproteinase-2 modulation by homocysteine.

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Liu, Tingjiao; Lin, Jinghan; Ju, Ting; Chu, Lei; Zhang, Liming

2015-08-01

Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metalloproteinase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (10, 100 or 200 μmol/L) or an MMP-2 inhibitor (10(-6) or 10(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, bone morphogenetic protein (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification proteins, and decreased anti-calcification protein levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification.

Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth.

Science.gov (United States)

Gloux, Karine; Leclerc, Marion; Iliozer, Harout; L'Haridon, René; Manichanh, Chaysavanh; Corthier, Gérard; Nalin, Renaud; Blottière, Hervé M; Doré, Joël

2007-06-01

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits.

Science.gov (United States)

Das, Suprem R; Uz, Metin; Ding, Shaowei; Lentner, Matthew T; Hondred, John A; Cargill, Allison A; Sakaguchi, Donald S; Mallapragada, Surya; Claussen, Jonathan C

2017-04-01

Graphene-based materials (GBMs) have displayed tremendous promise for use as neurointerfacial substrates as they enable favorable adhesion, growth, proliferation, spreading, and migration of immobilized cells. This study reports the first case of the differentiation of mesenchymal stem cells (MSCs) into Schwann cell (SC)-like phenotypes through the application of electrical stimuli from a graphene-based electrode. Electrical differentiation of MSCs into SC-like phenotypes is carried out on a flexible, inkjet-printed graphene interdigitated electrode (IDE) circuit that is made highly conductive (sheet resistance electrically stimulated/treated (etMSCs) display significant enhanced cellular differentiation and paracrine activity above conventional chemical treatment strategies [≈85% of the etMSCs differentiated into SC-like phenotypes with ≈80 ng mL -1 of nerve growth factor (NGF) secretion vs. 75% and ≈55 ng mL -1 for chemically treated MSCs (ctMSCs)]. These results help pave the way for in vivo peripheral nerve regeneration where the flexible graphene electrodes could conform to the injury site and provide intimate electrical simulation for nerve cell regrowth. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Step-wise and punctuated genome evolution drive phenotype changes of tumor cells

International Nuclear Information System (INIS)

Stepanenko, Aleksei; Andreieva, Svitlana; Korets, Kateryna; Mykytenko, Dmytro; Huleyuk, Nataliya; Vassetzky, Yegor; Kavsan, Vadym

2015-01-01

Highlights: • There are the step-wise continuous and punctuated phases of cancer genome evolution. • The system stresses during the different phases may lead to very different responses. • Stable transfection of an empty vector can result in genome and phenotype changes. • Functions of a (trans)gene can be opposite/versatile in cells with different genomes. • Contextually, temozolomide can both promote and suppress tumor cell aggressiveness. - Abstract: The pattern of genome evolution can be divided into two phases: the step-wise continuous phase (step-wise clonal evolution, stable dominant clonal chromosome aberrations (CCAs), and low frequency of non-CCAs, NCCAs) and punctuated phase (marked by elevated NCCAs and transitional CCAs). Depending on the phase, system stresses (the diverse CIN promoting factors) may lead to the very different phenotype responses. To address the contribution of chromosome instability (CIN) to phenotype changes of tumor cells, we characterized CCAs/NCCAs of HeLa and HEK293 cells, and their derivatives after genotoxic stresses (a stable plasmid transfection, ectopic expression of cancer-associated CHI3L1 gene or treatment with temozolomide) by conventional cytogenetics, copy number alterations (CNAs) by array comparative genome hybridization, and phenotype changes by cell viability and soft agar assays. Transfection of either the empty vector pcDNA3.1 or pcDNA3.1-CHI3L1 into 293 cells initiated the punctuated genome changes. In contrast, HeLa-CHI3L1 cells demonstrated the step-wise genome changes. Increased CIN correlated with lower viability of 293-pcDNA3.1 cells but higher colony formation efficiency (CFE). Artificial CHI3L1 production in 293-CHI3L1 cells increased viability and further contributed to CFE. The opposite growth characteristics of 293-CHI3L1 and HeLa-CHI3L1 cells were revealed. The effect and function of a (trans)gene can be opposite and versatile in cells with different genetic network, which is defined by

Step-wise and punctuated genome evolution drive phenotype changes of tumor cells

Energy Technology Data Exchange (ETDEWEB)

Stepanenko, Aleksei, E-mail: a.a.stepanenko@gmail.com [Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03680 (Ukraine); Andreieva, Svitlana; Korets, Kateryna; Mykytenko, Dmytro [Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03680 (Ukraine); Huleyuk, Nataliya [Institute of Hereditary Pathology, National Academy of Medical Sciences of Ukraine, Lviv 79008 (Ukraine); Vassetzky, Yegor [CNRS UMR8126, Université Paris-Sud 11, Institut de Cancérologie Gustave Roussy, Villejuif 94805 (France); Kavsan, Vadym [Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03680 (Ukraine)

2015-01-15

Highlights: • There are the step-wise continuous and punctuated phases of cancer genome evolution. • The system stresses during the different phases may lead to very different responses. • Stable transfection of an empty vector can result in genome and phenotype changes. • Functions of a (trans)gene can be opposite/versatile in cells with different genomes. • Contextually, temozolomide can both promote and suppress tumor cell aggressiveness. - Abstract: The pattern of genome evolution can be divided into two phases: the step-wise continuous phase (step-wise clonal evolution, stable dominant clonal chromosome aberrations (CCAs), and low frequency of non-CCAs, NCCAs) and punctuated phase (marked by elevated NCCAs and transitional CCAs). Depending on the phase, system stresses (the diverse CIN promoting factors) may lead to the very different phenotype responses. To address the contribution of chromosome instability (CIN) to phenotype changes of tumor cells, we characterized CCAs/NCCAs of HeLa and HEK293 cells, and their derivatives after genotoxic stresses (a stable plasmid transfection, ectopic expression of cancer-associated CHI3L1 gene or treatment with temozolomide) by conventional cytogenetics, copy number alterations (CNAs) by array comparative genome hybridization, and phenotype changes by cell viability and soft agar assays. Transfection of either the empty vector pcDNA3.1 or pcDNA3.1-CHI3L1 into 293 cells initiated the punctuated genome changes. In contrast, HeLa-CHI3L1 cells demonstrated the step-wise genome changes. Increased CIN correlated with lower viability of 293-pcDNA3.1 cells but higher colony formation efficiency (CFE). Artificial CHI3L1 production in 293-CHI3L1 cells increased viability and further contributed to CFE. The opposite growth characteristics of 293-CHI3L1 and HeLa-CHI3L1 cells were revealed. The effect and function of a (trans)gene can be opposite and versatile in cells with different genetic network, which is defined by

Development of a Functional Schwann Cell Phenotype from Autologous Porcine Bone Marrow Mononuclear Cells for Nerve Repair

Directory of Open Access Journals (Sweden)

Michael J. Rutten

2012-01-01

Full Text Available Adult bone marrow mononuclear cells (BM-MNCs are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6–8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF expression. Addition of neuregulin (1–25 nM increased p75(NGF levels at 24–48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca2+]i, with nucleotide potency being UTP=ATP>ADP>AMP>adenosine. Suramin blocked the ATP-induced [Ca2+]i but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca2+]i sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

Nicotine can skew the characterization of the macrophage type-1 (M{Phi}1) phenotype differentiated with granulocyte-macrophage colony-stimulating factor to the M{Phi}2 phenotype

Energy Technology Data Exchange (ETDEWEB)

Yanagita, Manabu; Kobayashi, Ryohei [Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, Osaka 565-0871 (Japan); Murakami, Shinya, E-mail: ipshinya@dent.osaka-u.ac.jp [Department of Periodontology, Division of Oral Biology and Disease Control, Osaka University Graduate School of Dentistry, Osaka 565-0871 (Japan)

2009-10-09

Macrophages (M{Phi}s) exhibit functional heterogeneity and plasticity in the local microenvironment. Recently, it was reported that M{Phi}s can be divided into proinflammatory M{Phi}s (M{Phi}1) and anti-inflammatory M{Phi}s (M{Phi}2) based on their polarized functional properties. Here, we report that nicotine, the major ingredient of cigarette smoke, can modulate the characteristics of M{Phi}1. Granulocyte-macrophage colony-stimulating factor-driven M{Phi}1 with nicotine (Ni-M{Phi}1) showed the phenotypic characteristics of M{Phi}2. Like M{Phi}2, Ni-M{Phi}1 exhibited antigen-uptake activities. Ni-M{Phi}1 suppressed IL-12, but maintained IL-10 and produced high amounts of MCP-1 upon lipopolysaccharide stimulation compared with M{Phi}1. Moreover, we observed strong proliferative responses of T cells to lipopolysaccharide-stimulated M{Phi}1, whereas Ni-M{Phi}1 reduced T cell proliferation and inhibited IFN-{gamma} production by T cells. These results suggest that nicotine can change the functional characteristics of M{Phi} and skew the M{Phi}1 phenotype to M{Phi}2. We propose that nicotine is a potent regulator that modulates immune responses in microenvironments.

Strategy of metabolic phenotype modulation in Portunus trituberculatus exposed to low salinity.

Science.gov (United States)

Ye, Yangfang; An, Yanpeng; Li, Ronghua; Mu, Changkao; Wang, Chunlin

2014-04-16

Extreme low salinity influences normal crab growth, morphogenesis, and production. Some individuals of swimming crab Portunus trituberculatus have, however, an inherent ability to adapt to such a salinity fluctuation. This study investigated the dynamic metabolite alterations of two P. trituberculatus strains, namely, a wild one and a screened (low-salinity tolerant) one in response to low-salinity challenge by combined use of NMR spectroscopy and high-throughput data analysis. The dominant metabolites in crab muscle were found to comprise amino acids, sugars, carboxylic acids, betaine, trimethylamine-N-oxide, 2-pyridinemethanol, trigonelline, and nucleotides. These results further showed that the strategy of metabolic modulation of P. trituberculatus after low-salinity stimulus includes osmotic rebalancing, enhanced gluconeogenesis from amino acids, and energy accumulation. These metabolic adaptations were manifested in the accumulation of trimethylamine-N-oxide, ATP, 2-pyridinemethanol, and trigonelline and in the depletion of the amino acid pool as well as in the fluctuation of inosine levels. This lends support to the fact that the low-salinity training accelerates the responses of crabs to low-salinity stress. These findings provide a comprehensive insight into the mechanisms of metabolic modulation in P. trituberculatus in response to low salinity. This work highlights the approach of NMR-based metabonomics in conjunction with multivariate data analysis and univariate data analysis in understanding the strategy of metabolic phenotype modulation against stressors.

Glioma Cells in the Tumor Periphery Have a Stem Cell Phenotype

DEFF Research Database (Denmark)

Munthe, Sune; Petterson, Stine Asferg; Dahlrot, Rikke Hedegaard

2016-01-01

and a panel of markers was used. The panel comprised of six stem cell-related markers (CD133, Musashi-1, Bmi-1, Sox-2, Nestin and Glut-3), a proliferation marker (Ki-67) as well as a chemo-resistance marker (MGMT). Computer-based automated classifiers were designed to measure the mIDH1 positive nucleus area......-fraction of the chosen markers. Moreover, orthotopic glioblastoma xenografts from five different patient-derived spheroid cultures were obtained and the tumor cells identified by human specific immunohistochemical markers. The results showed that tumor cells in the periphery of patient gliomas expressed stem cell...... in the periphery of patient gliomas have a stem cell phenotype, although it is less pronounced than in the tumor core. Novel therapies aiming at preventing recurrence should therefore take tumor stemness into account. Migrating cells in orthotopic glioblastoma xenografts preserve expression and stem cell markers...

When stem cells grow old: phenotypes and mechanisms of stem cell aging

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Schultz, Michael B.; Sinclair, David A.

2016-01-01

All multicellular organisms undergo a decline in tissue and organ function as they age. An attractive theory is that a loss in stem cell number and/or activity over time causes this decline. In accordance with this theory, aging phenotypes have been described for stem cells of multiple tissues, including those of the hematopoietic system, intestine, muscle, brain, skin and germline. Here, we discuss recent advances in our understanding of why adult stem cells age and how this aging impacts diseases and lifespan. With this increased understanding, it is feasible to design and test interventions that delay stem cell aging and improve both health and lifespan. PMID:26732838

Sickle cell disease clinical phenotypes in children from South ...

African Journals Online (AJOL)

2014-07-20

Jul 20, 2014 ... Background:The clinical phenotypes of children with sickle cell disease (SCD) are poorly described in many sub-Saharan countries ..... World Health Organization. ... apps.who.int/gb/ebwha/pdf_files/WHA59/A59_9‑en.pdf.

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

DEFF Research Database (Denmark)

Martens, Geert A; Jiang, Lei; Hellemans, Karine H

2011-01-01

The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

Increased memory phenotypes of CD4+ and CD8+ T cells in ...

African Journals Online (AJOL)

Leonard Mboera

Tanzania Journal of Health Research ... In this study, we investigated the phenotype and activation ... Children with Sickle Cell Anaemia (SCA), the homozygous form of Sickle Cell ..... J.L., Fenton, P., Blumberg, N. & Walters, M.C. (2005) Hematopoietic stem cell ... cell anaemia patients and effects of hydroxyurea therapy.

Physical constraints in cell fate specification. A case in point: Microgravity and phenotypes differentiation.

Science.gov (United States)

Masiello, Maria Grazia; Verna, Roberto; Cucina, Alessandra; Bizzarri, Mariano

2018-05-01

Data obtained by studying mammalian cells in absence of gravity strongly support the notion that cell fate specification cannot be understood according to the current molecular model. A paradigmatic case in point is provided by studying cell populations growing in absence of gravity. When the physical constraint (gravity) is 'experimentally removed', cells spontaneously allocate into two morphologically different phenotypes. Such phenomenon is likely enacted by the intrinsic stochasticity, which, in turn, is successively 'canalized' by a specific gene regulatory network. Both phenotypes are thermodynamically and functionally 'compatibles' with the new, modified environment. However, when the two cell subsets are reseeded into the 1g gravity field the two phenotypes collapse into one. Gravity constraints the system in adopting only one phenotype, not by selecting a pre-existing configuration, but more precisely shaping it de-novo through the modification of the cytoskeleton three-dimensional structure. Overall, those findings highlight how macro-scale features are irreducible to lower-scale explanations. The identification of macroscale control parameters - as those depending on the field (gravity, electromagnetic fields) or emerging from the cooperativity among the field's components (tissue stiffness, cell-to-cell connectivity) - are mandatory for assessing boundary conditions for models at lower scales, thus providing a concrete instantiation of top-down effects. Copyright © 2018 Elsevier Ltd. All rights reserved.

Agmatine Modulates the Phenotype of Macrophage Acute Phase after Spinal Cord Injury in Rats.

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Kim, Jae Hwan; Kim, Jae Young; Mun, Chin Hee; Suh, Minah; Lee, Jong Eun

2017-10-01

Agmatine is a decarboxylated arginine by arginine decarboxylase. Agmatine is known to be a neuroprotective agent. It has been reported that agmatine works as a NMDA receptor blocker or a competitive nitric oxide synthase inhibitor in CNS injuries. In spinal cord injury, agmatine showed reduction of neuropathic pain, improvement of locomotor function, and neuroprotection. Macrophage is a key cellular component in neuroinflammation, a major cause of impairment after spinal cord injury. Macrophage has subtypes, M1 and M2 macrophages. M1 macrophage induces a pro-inflammatory response, but M2 inspires an anti-inflammatory response. In this study, it was clarified whether the neuroprotective effect of agmatine is related with the modulation of macrophage subdivision after spinal cord injury. Spinal cord injury was induced in rats with contusion using MASCIS. Animals received agmatine (100 mg/kg, IP) daily for 6 days beginning the day after spinal cord injury. The proportion of M1 and M2 macrophages are confirmed with immunohistochemistry and FACS. CD206 + & ED1 + cells were counted as M2 macrophages. The systemic treatment of agmatine increased M2 macrophages caudal side to epicenter 1 week after spinal cord injury in immunohistochemistry. M2 macrophage related markers, Arginase-1 and CD206 mRNA, were increased in the agmatine treatment group and M2 macrophage expressing and stimulated cytokine, IL-10 mRNA, also was significantly overexpressed by agmatine injection. Among BMPs, BMP2/4/7, agmatine significantly increased only the expression of BMP2 known to reduce M1 macrophage under inflammatory status. These results suggest that agmatine reduces impairment after spinal cord injury through modulating the macrophage phenotype.

T-bet and Eomes are differentially linked to the exhausted phenotype of CD8+ T cells in HIV infection.

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Marcus Buggert

2014-07-01

Full Text Available CD8(+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8(+ T cell differentiation, T-bet and Eomesodermin (Eomes, have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4, functional characteristics and memory differentiation of CD8(+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8(+ T cells was elevated in chronically infected individuals and highly associated with a T-bet(dimEomes(hi expressional profile. Interestingly, both resting and activated HIV-specific CD8(+ T cells in chronic infection were almost exclusively T-bet(dimEomes(hi cells, while CMV-specific CD8(+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-bet(dimEomes(hi virus-specific CD8(+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector characteristics. The transitional and exhausted phenotype of HIV-specific CD8(+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8(+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8(+ T cells to control the viral replication post-ART cessation.

Concentration characteristics and cell arrangement in luminescent concentrator PV modules; Keiko shukogata taiyo denchi module no cell haichi to shuko tokusei

Energy Technology Data Exchange (ETDEWEB)

Inamura, A [Science University of Tokyo, Tokyo (Japan); Sakuta, K [Electrotechnical Laboratory, Tsukuba (Japan)

1997-11-25

A luminescent concentrator PV module requires no tracking equipment and can use scattered light. A mini PV module was prepared from a luminescent plate of 100times100times3mm, and a single-crystalline PV cell of 100times20mm. Characteristics of various prototype modules with different PV cell areas and cell arrangements were also measured. Four kinds of edge reflecting materials with different reflectances by various white coating were applied to Al sashes for module frames, and each sash was fixed on one edge of the luminescent plate. In experiment, 3 other edges were covered with black tapes to reduce each reflectance to 0%. Although PV module output was affected by reflectance of edges, the output was satisfactory at 90% or more in reflectance showing no difference in output. A concentrating efficiency decreased with an increase in luminescent plate (concentrator) area, while it was improved by cell arrangement with short optical pass length, and cell arrangement hardly affected by edge reflection. 4 refs., 7 figs.

Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

International Nuclear Information System (INIS)

Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

2005-01-01

Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

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Katarzyna Wachowicz

Full Text Available Protein kinase C θ (PKCθ is involved in signaling downstream of the T cell antigen receptor (TCR and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures and in vivo (experimental autoimmune encephalomyelitis model, EAE techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt, accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ-/- CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.

The therapeutic implications of plasticity of the cancer stem cell phenotype.

Directory of Open Access Journals (Sweden)

Kevin Leder

2010-12-01

Full Text Available The cancer stem cell hypothesis suggests that tumors contain a small population of cancer cells that have the ability to undergo symmetric self-renewing cell division. In tumors that follow this model, cancer stem cells produce various kinds of specified precursors that divide a limited number of times before terminally differentiating or undergoing apoptosis. As cells within the tumor mature, they become progressively more restricted in the cell types to which they can give rise. However, in some tumor types, the presence of certain extra- or intracellular signals can induce committed cancer progenitors to revert to a multipotential cancer stem cell state. In this paper, we design a novel mathematical model to investigate the dynamics of tumor progression in such situations, and study the implications of a reversible cancer stem cell phenotype for therapeutic interventions. We find that higher levels of dedifferentiation substantially reduce the effectiveness of therapy directed at cancer stem cells by leading to higher rates of resistance. We conclude that plasticity of the cancer stem cell phenotype is an important determinant of the prognosis of tumors. This model represents the first mathematical investigation of this tumor trait and contributes to a quantitative understanding of cancer.

When stem cells grow old: phenotypes and mechanisms of stem cell aging.

Science.gov (United States)

Schultz, Michael B; Sinclair, David A

2016-01-01

All multicellular organisms undergo a decline in tissue and organ function as they age. An attractive theory is that a loss in stem cell number and/or activity over time causes this decline. In accordance with this theory, aging phenotypes have been described for stem cells of multiple tissues, including those of the hematopoietic system, intestine, muscle, brain, skin and germline. Here, we discuss recent advances in our understanding of why adult stem cells age and how this aging impacts diseases and lifespan. With this increased understanding, it is feasible to design and test interventions that delay stem cell aging and improve both health and lifespan. © 2016. Published by The Company of Biologists Ltd.

In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

Science.gov (United States)

Reyes-Aguirre, Luis I; Quintero, Heberto; Estrada-Leyva, Brenda; Lamas, Mónica

2018-01-01

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

Module-based complexity formation: periodic patterning in feathers and hairs.

Science.gov (United States)

Chuong, Cheng-Ming; Yeh, Chao-Yuan; Jiang, Ting-Xin; Widelitz, Randall

2013-01-01

Patterns describe order which emerges from homogeneity. Complex patterns on the integument are striking because of their visibility throughout an organism’s lifespan. Periodic patterning is an effective design because the ensemble of hair or feather follicles (modules) allows the generation of complexity, including regional variations and cyclic regeneration, giving the skin appendages a new lease on life. Spatial patterns include the arrangements of feathers and hairs in specific number, size, and spacing.We explorehowa field of equivalent progenitor cells can generate periodically arranged modules based on genetic information, physical–chemical rules and developmental timing. Reconstitution experiments suggest a competitive equilibrium regulated by activators/inhibitors involving Turing reaction-diffusion. Temporal patterns result from oscillating stem cell activities within each module (microenvironment regulation), reflected as growth (anagen) and resting (telogen) phases during the cycling of feather and hair follicles. Stimulating modules with activators initiates the spread of regenerative hair waves, while global inhibitors outside each module (macroenvironment) prevent this. Different wave patterns can be simulated by cellular automata principles. Hormonal status and seasonal changes can modulate appendage phenotypes, leading to ‘organ metamorphosis’, with multiple ectodermal organ phenotypes generated from the same precursors. We discuss potential novel evolutionary steps using this module-based complexity in several amniote integument organs, exemplified by the spectacular peacock feather pattern. We thus explore the application of the acquired knowledge of patterning in tissue engineering. New hair follicles can be generated after wounding. Hairs and feathers can be reconstituted through self-organization of dissociated progenitor cells. © 2012 Wiley Periodicals, Inc.

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces

International Nuclear Information System (INIS)

Lee, Chung-Ho; Kim, Youn-Jeong; Jang, Je-Hee; Park, Jin-Woo

2016-01-01

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function. (paper)

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces

Science.gov (United States)

Lee, Chung-Ho; Kim, Youn-Jeong; Jang, Je-Hee; Park, Jin-Woo

2016-02-01

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function.

Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines

International Nuclear Information System (INIS)

Martowicz, Agnieszka; Spizzo, Gilbert; Gastl, Guenther; Untergasser, Gerold

2012-01-01

The epithelial cell adhesion molecule (EpCAM) has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype. For this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAM high breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3). Moreover, EpCAM low breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t) and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth. In comparison to non-specific silencing controls (n/s-crtl) knockdown of EpCAM (E#2) in EpCAM high cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAM low cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes. The role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer

A platform for high-throughput bioenergy production phenotype characterization in single cells

Science.gov (United States)

Kelbauskas, Laimonas; Glenn, Honor; Anderson, Clifford; Messner, Jacob; Lee, Kristen B.; Song, Ganquan; Houkal, Jeff; Su, Fengyu; Zhang, Liqiang; Tian, Yanqing; Wang, Hong; Bussey, Kimberly; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers. PMID:28349963

CYBRD1 as a modifier gene that modulates iron phenotype in HFE p.C282Y homozygous patients.

Science.gov (United States)

Pelucchi, Sara; Mariani, Raffaella; Calza, Stefano; Fracanzani, Anna Ludovica; Modignani, Giulia Litta; Bertola, Francesca; Busti, Fabiana; Trombini, Paola; Fraquelli, Mirella; Forni, Gian Luca; Girelli, Domenico; Fargion, Silvia; Specchia, Claudia; Piperno, Alberto

2012-12-01

Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors. In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption. We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409. While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on

Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

Science.gov (United States)

Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

2016-06-02

After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

Science.gov (United States)

Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

2018-03-01

Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

Temperature dependence of photovoltaic cells, modules, and systems

Energy Technology Data Exchange (ETDEWEB)

Emery, K.; Burdick, J.; Caiyem, Y. [National Renewable Energy Lab., Golden, CO (United States)] [and others

1996-05-01

Photovoltaic (PV) cells and modules are often rated in terms of a set of standard reporting conditions defined by a temperature, spectral irradiance, and total irradiance. Because PV devices operates over a wide range of temperatures and irradiances, the temperature and irradiance related behavior must be known. This paper surveys the temperature dependence of crystalline and thin-film, state-of-the-art, research-size cells, modules, and systems measured by a variety of methods. The various error sources and measurement methods that contribute to cause differences in the temperature coefficient for a given cell or module measured with various methods are discussed.

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

International Nuclear Information System (INIS)

Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

2011-01-01

Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

Science.gov (United States)

Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

Compatibility of copper-electroplated cells with Metal Wrap Through module materials

Energy Technology Data Exchange (ETDEWEB)

Bennett, I.J.; Geerligs, L.J.; Olson, C.L.; Goris, M.J.A.A. [ECN Solar Energy, Petten (Netherlands)

2013-10-16

As part of the European FP7 RandD project 'Cu-PV', the compatibility of copper-electroplated metal wrapthrough (MWT) cells with conductive adhesives has been investigated. The objectives of this project include to reduce, by the use of copper plating, the amount of silver utilized in cell manufacturing, and to demonstrate the compatibility of high-power n-type back-contact module technology with copper-plated cells. The overall goal is to reduce the impact on the environment of cell and module manufacture. MWT module technology as developed by ECN uses conductive adhesive to make the interconnection between cells and a conductive backsheet foil. These adhesives have been proved to result in very reliable modules in the case of cells with fired silver metallization. To determine the compatibility of conductive adhesive with copper-plated cells, component tests were performed, followed by the manufacture of modules with copperplated cells and conductive adhesive interconnections. Climate chamber testing of these modules showed that the adhesive is compatible with the copper-plated cells. The next steps include further optimization of the plating process and additional testing at the module level.

Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells

DEFF Research Database (Denmark)

Fornara, O; Bartek, J; Rahbar, A

2016-01-01

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express...... human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary...... GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co...

Comparison Of Liver Cell Models Using The Basel Phenotyping Cocktail

Directory of Open Access Journals (Sweden)

Benjamin Berger

2016-11-01

Full Text Available Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH cultures. We investigated the suit-ability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4] in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6 and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least 4-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20µM rifampicin, mRNA increased 3 to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6 and 17-fold (CYP3A4 for 2D-PHH and 4-fold (CYP3A4 for HepG2. In 3D-PHH at least a 2-fold in-crease in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model.Our studies show that 3D-PHH and (with some limitations HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to as-sess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro.

p-Type MWT. Integrated cell and module technology

Energy Technology Data Exchange (ETDEWEB)

Tool, C.J.J.; Kossen, E.J.; Bennett, I.J.

2013-10-15

A major issue of concern in MWT solar cells is the increased leakage current at reversed bias voltage through the vias compared. At ECN we have been working on reducing this leakage current to levels comparable to H-pattern cells. In this study we present the results of this work. We further show the benefit of a combined cell and module design for MWT solar cells. At the cell level, MWT production costs per wafer are comparable with H-pattern while the cell output increases. At the module level this design results in a further increase of the power output.

p-type MWT. Integrated Cell and Module Technology

Energy Technology Data Exchange (ETDEWEB)

Tool, C.J.J.; Kossen, E.J.; Bennett, I.J. [ECN Solar Energy, Petten (Netherlands)

2013-03-15

A major issue of concern in MWT (metal wrap-through) solar cells is the increased leakage current at reversed bias voltage through the vias compared. At ECN we have been working on reducing this leakage current to levels comparable to H-pattern cells. In this study we present the results of this work. We further show the benefit of a combined cell and module design for MWT solar cells. At the cell level, MWT production costs per wafer are comparable with H-pattern while the cell output increases. At the module level this design results in a further increase of the power output.

Phenotypic and Functional Properties of Tumor-Infiltrating Regulatory T Cells

Directory of Open Access Journals (Sweden)

Gap Ryol Lee

2017-01-01

Full Text Available Regulatory T (Treg cells maintain immune homeostasis by suppressing excessive immune responses. Treg cells induce tolerance against self- and foreign antigens, thus preventing autoimmunity, allergy, graft rejection, and fetus rejection during pregnancy. However, Treg cells also infiltrate into tumors and inhibit antitumor immune responses, thus inhibiting anticancer therapy. Depleting whole Treg cell populations in the body to enhance anticancer treatments will produce deleterious autoimmune diseases. Therefore, understanding the precise nature of tumor-infiltrating Treg cells is essential for effectively targeting Treg cells in tumors. This review summarizes recent results relating to Treg cells in the tumor microenvironment, with particular emphasis on their accumulation, phenotypic, and functional properties, and targeting to enhance the efficacy of anticancer treatment.

Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

International Nuclear Information System (INIS)

Yang, Qiwei; Tian, Yufeng; Ostler, Kelly R; Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Godley, Lucy A; Cohn, Susan L

2010-01-01

Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation

Increased entropy of signal transduction in the cancer metastasis phenotype

Directory of Open Access Journals (Sweden)

Teschendorff Andrew E

2010-07-01

Full Text Available Abstract Background The statistical study of biological networks has led to important novel biological insights, such as the presence of hubs and hierarchical modularity. There is also a growing interest in studying the statistical properties of networks in the context of cancer genomics. However, relatively little is known as to what network features differ between the cancer and normal cell physiologies, or between different cancer cell phenotypes. Results Based on the observation that frequent genomic alterations underlie a more aggressive cancer phenotype, we asked if such an effect could be detectable as an increase in the randomness of local gene expression patterns. Using a breast cancer gene expression data set and a model network of protein interactions we derive constrained weighted networks defined by a stochastic information flux matrix reflecting expression correlations between interacting proteins. Based on this stochastic matrix we propose and compute an entropy measure that quantifies the degree of randomness in the local pattern of information flux around single genes. By comparing the local entropies in the non-metastatic versus metastatic breast cancer networks, we here show that breast cancers that metastasize are characterised by a small yet significant increase in the degree of randomness of local expression patterns. We validate this result in three additional breast cancer expression data sets and demonstrate that local entropy better characterises the metastatic phenotype than other non-entropy based measures. We show that increases in entropy can be used to identify genes and signalling pathways implicated in breast cancer metastasis and provide examples of de-novo discoveries of gene modules with known roles in apoptosis, immune-mediated tumour suppression, cell-cycle and tumour invasion. Importantly, we also identify a novel gene module within the insulin growth factor signalling pathway, alteration of which may

Hsp90 selectively modulates phenotype in vertebrate development.

Directory of Open Access Journals (Sweden)

Patricia L Yeyati

2007-03-01

Full Text Available Compromised heat shock protein 90 (Hsp90 function reveals cryptic phenotypes in flies and plants. These observations were interpreted to suggest that this molecular stress-response chaperone has a capacity to buffer underlying genetic variation. Conversely, the protective role of Hsp90 could account for the variable penetrance or severity of some heritable developmental malformations in vertebrates. Using zebrafish as a model, we defined Hsp90 inhibitor levels that did not induce a heat shock response or perturb phenotype in wild-type strains. Under these conditions the severity of the recessive eye phenotype in sunrise, caused by a pax6b mutation, was increased, while in dreumes, caused by a sufu mutation, it was decreased. In another strain, a previously unobserved spectrum of severe structural eye malformations, reminiscent of anophthalmia, microphthalmia, and nanophthalmia complex in humans, was uncovered by this limited inhibition of Hsp90 function. Inbreeding of offspring from selected unaffected carrier parents led to significantly elevated malformation frequencies and revealed the oligogenic nature of this phenotype. Unlike in Drosophila, Hsp90 inhibition can decrease developmental stability in zebrafish, as indicated by increased asymmetric presentation of anophthalmia, microphthalmia, and nanophthalmia and sunrise phenotypes. Analysis of the sunrise pax6b mutation suggests a molecular mechanism for the buffering of mutations by Hsp90. The zebrafish studies imply that mild perturbation of Hsp90 function at critical developmental stages may underpin the variable penetrance and expressivity of many developmental anomalies where the interaction between genotype and environment plays a major role.

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

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Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

2012-01-01

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

Science.gov (United States)

Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M; Iorio, Ronald M; Li, Jianrong

2012-11-01

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures.

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Tom Bongiorno

Full Text Available The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of "soft" cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.

Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

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Devaveena Dey

Full Text Available BACKGROUND: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. METHODOLOGY: Single cell suspensions derived from human breast 'organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. PRINCIPAL FINDINGS: We show that primary mammospheres contain a distinct side-population (SP that displays a CD24(low/CD44(low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high/CD24(low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1 mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. CONCLUSIONS: Thus, the self-renewal potential of human breast stem cells is

Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment

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Hasan, Al Shaimaa; Luo, Lan; Yan, Chen; Zhang, Tian-Xia; Urata, Yoshishige; Goto, Shinji; Mangoura, Safwat A.; Abdel-Raheem, Mahmoud H.; Zhang, Shouhua; Li, Tao-Sheng

2016-01-01

Cardiosphere-derived cells (CDCs), one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1) into a regulatory anti-inflammatory phenotype (M2). Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages), but decreased the expression of CD86 (a marker for M1 macrophages) 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair. PMID:27764217

Cell-Intrinsic Roles for Autophagy in Modulating CD4 T Cell Functions

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Elise Jacquin

2018-05-01

Full Text Available The catabolic process of autophagy plays important functions in inflammatory and immune responses by modulating innate immunity and adaptive immunity. Over the last decade, a cell-intrinsic role for autophagy in modulating CD4 T cell functions and differentiation was revealed. After the initial observation of autophagosomes in effector CD4 T cells, further work has shown that not only autophagy levels are modulated in CD4 T cells in response to environmental signals but also that autophagy critically affects the biology of these cells. Mouse models of autophagy deletion in CD4 T cells have indeed shown that autophagy is essential for CD4 T cell survival and homeostasis in peripheral lymphoid organs. Furthermore, autophagy is required for CD4 T cell proliferation and cytokine production in response to T cell receptor activation. Recent developments have uncovered that autophagy controls CD4 T cell differentiation and functions. While autophagy is required for the maintenance of immunosuppressive functions of regulatory T cells, it restrains the differentiation of TH9 effector cells, thus limiting their antitumor and pro-inflammatory properties. We will here discuss these findings that collectively suggest that therapeutic strategies targeting autophagy could be exploited for the treatment of cancer and inflammatory diseases.

Circuit analysis method for thin-film solar cell modules

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Burger, D. R.

1985-01-01

The design of a thin-film solar cell module is dependent on the probability of occurrence of pinhole shunt defects. Using known or assumed defect density data, dichotomous population statistics can be used to calculate the number of defects expected in a module. Probability theory is then used to assign the defective cells to individual strings in a selected series-parallel circuit design. Iterative numerical calculation is used to calcuate I-V curves using cell test values or assumed defective cell values as inputs. Good and shunted cell I-V curves are added to determine the module output power and I-V curve. Different levels of shunt resistance can be selected to model different defect levels.

Solar cell junction temperature measurement of PV module

KAUST Repository

Huang, B.J.; Yang, P.E.; Lin, Y.P.; Lin, B.Y.; Chen, H.J.; Lai, R.C.; Cheng, J.S.

2011-01-01

The present study develops a simple non-destructive method to measure the solar cell junction temperature of PV module. The PV module was put in the environmental chamber with precise temperature control to keep the solar PV module as well

SAP is required for the development of innate phenotype in H2-M3-restricted CD8+ T cells1

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Bediako, Yaw; Bian, Yao; Zhang, Hong; Cho, Hoonsik; Stein, Paul L.; Wang, Chyung-Ru

2012-01-01

H2-M3-restricted T cells have a pre-activated surface phenotype, rapidly expand and produce cytokines upon stimulation and as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αβ co-receptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8+ T cells. Although iNKT cells are also innate lymphocytes, they are selected exclusively on hematopoietic cells (HC), while M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells (TEC). Moreover, their phenotypes differ depending on what cells mediate their selection. Though there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. SAP is required for the development of iNKT cells and mediates signals from SLAM receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models we demonstrate that while M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of pre-activated phenotype and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a pre-activated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection. PMID:23041566

76 FR 78313 - Crystalline Silicon Photovoltaic Cells and Modules From China

Science.gov (United States)

2011-12-16

...)] Crystalline Silicon Photovoltaic Cells and Modules From China Determinations On the basis of the record \\1... injured by reason of imports from China of crystalline silicon photovoltaic cells and modules, provided... imports of crystalline silicon photovoltaic cells and modules from China. Accordingly, effective October...

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation.

Science.gov (United States)

Ching, Carly; Gozzi, Kevin; Heinemann, Björn; Chai, Yunrong; Godoy, Veronica G

2017-06-01

In the nosocomial opportunistic pathogen Acinetobacter baumannii , RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii Here, we identify a structured 5' untranslated region (UTR) in the recA transcript which serves as a cis -regulatory element. We show that a predicted stem-loop structure in this 5' UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5' UTR influences intracellular RecA protein levels and, in vivo , impairing the formation of the stem-loop structure of the recA 5' UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5' UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen. IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis -acting 5' UTR regulates recA transcript stability, RecA protein levels, and DNA

MODULATING EFFECT OF THE −158 GΓ (C→T XMN-1 POLYMORPHISM IN INDIAN SICKLE CELL PATIENTS

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Sanjay Pandey

2012-01-01

Full Text Available Xmn-1 polymorphism is a known factor, which increases fetal haemoglobin production. Among the inherited disorders of blood, thalassaemia and SCD constitutes a major bulk of genetic diseases in India.  Our aim was to verify the role of the Xmn I polymorphism as a modulating factor in sickle cell patients and frequency of the polymorphism in Indian sickle cell patients. Subjects were 60 sickles homozygous and 75 sickle beta thalassemia patients. 5 ml blood   samples collected from patients. Screening of sickle patients done by HPLC. An automated cell analyzer SYSMEX (K-4500 Model used to analyze the CBC of patients.Xmn1 polymorphism analysis done by PCR-RFLP and Statistical analysis was performed on GraphPad static’s software. t test applied to compare the means amongst group. Among the sickle homozygous 27 were   heterozygous (+/- and 19 were   homozygous (+/+ while 30 were heterozygous and 24 were homozygous in sickle β-thalassemia patients. Extremely significant differences (p-value <0.001 of hematological parameters seen among patient with xmn-1 carrier and without the xmn-1 carrier. In our cases the clinical symptom less frequent and higher HbF level with Xmn-1 carriers. Presence of Xmn-1 polymorphism in sickle patients with higher HbF that improve phenotypic presentation in the sickle cell patients. We conclude that the phenotype of Indian sickle cell patients influenced by Xmn-1 polymorphism.

Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

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Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason; Ng, Cherie T; Krogan, Nevan; Sharan, Roded; Ideker, Trey

2016-02-24

Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

Giant cell lesions with a Noonan-like phenotype: a case report.

Science.gov (United States)

Cancino, Claudia Marcela H; Gaião, Léonilson; Sant'Ana Filho, Manoel; Oliveira, Flavio Augusto Marsiaj

2007-05-01

The purpose of this article is to describe a case of multiple giant cell lesions of the mandible that occurred in a 14-year-old girl with phenotypic characteristics associated with Noonan Syndrome (NS). NS is a dysmorphic disorder characterized by hypertelorism, short stature, congenital heart defects, short and webbed neck, skeletal anomalies, and bleeding diathesis. A 14-year-old girl with a previous diagnosis of NS (sporadic case) presented with multiple radiolucent lesions in the body and ramus of her mandible. In terms of clinical behavior and the described radiographic characteristics, giant cells lesions with Noonan-like phenotype can be considered a form of cherubism. Therefore, surgical intervention is not necessary, but radiographic follow-up and observation is very important during the control and gradual regression of the lesions.

77 FR 72884 - Crystalline Silicon Photovoltaic Cells and Modules From China

Science.gov (United States)

2012-12-06

... Silicon Photovoltaic Cells and Modules From China Determinations On the basis of the record \\1\\ developed... imports of crystalline silicon photovoltaic cells and modules from China, provided for in subheadings 8501... silicon photovoltaic cells and modules from China. Chairman Irving A. Williamson and Commissioner Dean A...

Gigantol Suppresses Cancer Stem Cell-Like Phenotypes in Lung Cancer Cells

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Narumol Bhummaphan

2015-01-01

Full Text Available As cancer stem cells (CSCs contribute to malignancy, metastasis, and relapse of cancers, potential of compound in inhibition of CSCs has garnered most attention in the cancer research as well as drug development fields recently. Herein, we have demonstrated for the first time that gigantol, a pure compound isolated from Dendrobium draconis, dramatically suppressed stem-like phenotypes of human lung cancer cells. Gigantol at nontoxic concentrations significantly reduced anchorage-independent growth and survival of the cancer cells. Importantly, gigantol significantly reduced the ability of the cancer cells to form tumor spheroids, a critical hallmark of CSCs. Concomitantly, the treatment of the compound was shown to reduce well-known lung CSCs markers, including CD133 and ALDH1A1. Moreover, we revealed that gigantol decreased stemness in the cancer cells by suppressing the activation of protein kinase B (Akt signal which in turn decreased the cellular levels of pluripotency and self-renewal factors Oct4 and Nanog. In conclusion, gigantol possesses CSCs suppressing activity which may facilitate the development of this compound for therapeutic approaches by targeting CSCs.

Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

Science.gov (United States)

Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

2016-01-01

Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

Characterization of cell mismatch in a multi-crystalline silicon photovoltaic module

International Nuclear Information System (INIS)

Crozier, J.L.; Dyk, E.E. van; Vorster, F.J.

2012-01-01

In this study the causes and effects of cell mismatch were identified in a multi-crystalline silicon photovoltaic module. Different techniques were used to identify the causes of the mismatch, including Electroluminescence (EL) imaging, Infrared (IR) imaging, current–voltage (I–V) characteristics, worst-case cell determination and Large Area Laser Beam Induced Current (LA-LBIC) scans. In EL images the cracked cells, broken fingers and material defects are visible. The presence of poorly contacted cells results in the formation of hot-spots. LA-LBIC line scans give the relative photoresponse of the cells in the module. However, this technique is limited due to the penetration depth of the laser beam. The worst case cell determination compares the I–V curves of the whole module with the I–V curve of the module with one cell covered, allowing the evaluation of the performance of each cell in a series-connected string. These methods allowed detection of the poorly performing cells in the module. Using all these techniques an overall view of the photoresponse in the cells and their performance is obtained.

Characterization of cell mismatch in a multi-crystalline silicon photovoltaic module

Energy Technology Data Exchange (ETDEWEB)

Crozier, J.L., E-mail: s207094248@live.nmmu.ac.za [Department of Physics, P.O. Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth 6031 (South Africa); Dyk, E.E. van; Vorster, F.J. [Department of Physics, P.O. Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth 6031 (South Africa)

2012-05-15

In this study the causes and effects of cell mismatch were identified in a multi-crystalline silicon photovoltaic module. Different techniques were used to identify the causes of the mismatch, including Electroluminescence (EL) imaging, Infrared (IR) imaging, current-voltage (I-V) characteristics, worst-case cell determination and Large Area Laser Beam Induced Current (LA-LBIC) scans. In EL images the cracked cells, broken fingers and material defects are visible. The presence of poorly contacted cells results in the formation of hot-spots. LA-LBIC line scans give the relative photoresponse of the cells in the module. However, this technique is limited due to the penetration depth of the laser beam. The worst case cell determination compares the I-V curves of the whole module with the I-V curve of the module with one cell covered, allowing the evaluation of the performance of each cell in a series-connected string. These methods allowed detection of the poorly performing cells in the module. Using all these techniques an overall view of the photoresponse in the cells and their performance is obtained.

Chitosan Feasibility to Retain Retinal Stem Cell Phenotype and Slow Proliferation for Retinal Transplantation

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Girish K. Srivastava

2014-01-01

Full Text Available Retinal stem cells (RSCs are promising in cell replacement strategies for retinal diseases. RSCs can migrate, differentiate, and integrate into retina. However, RSCs transplantation needs an adequate support; chitosan membrane (ChM could be one, which can carry RSCs with high feasibility to support their integration into retina. RSCs were isolated, evaluated for phenotype, and subsequently grown on sterilized ChM and polystyrene surface for 8 hours, 1, 4, and 11 days for analysing cell adhesion, proliferation, viability, and phenotype. Isolated RSCs expressed GFAP, PKC, isolectin, recoverin, RPE65, PAX-6, cytokeratin 8/18, and nestin proteins. They adhered (28 ± 16%, 8 hours and proliferated (40 ± 20 cells/field, day 1 and 244 ± 100 cells/field, day 4 significantly low (P95% and phenotype (cytokeratin 8/18, PAX6, and nestin proteins expression, day 11 on both surfaces (ChM and polystyrene. RSCs did not express alpha-SMA protein on both surfaces. RSCs express proteins belonging to epithelial, glial, and neural cells, confirming that they need further stimulus to reach a final destination of differentiation that could be provided in in vivo condition. ChM does not alternate RSCs behaviour and therefore can be used as a cell carrier so that slow proliferating RSCs can migrate and integrate into retina.

U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells

International Nuclear Information System (INIS)

Torsvik, Anja; Stieber, Daniel; Enger, Per Øyvind; Golebiewska, Anna; Molven, Anders; Svendsen, Agnete; Westermark, Bengt; Niclou, Simone P; Olsen, Thale Kristin; Chekenya Enger, Martha; Bjerkvig, Rolf

2014-01-01

It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line

A Neutrophil Phenotype Model for Extracorporeal Treatment of Sepsis.

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Alexander D Malkin

2015-10-01

Full Text Available Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage in sepsis. Interleukin-8 (IL-8, a key modulator of neutrophil function, signals through neutrophil specific surface receptors CXCR-1 and CXCR-2. In this study a mechanistic computational model was used to evaluate and deploy an extracorporeal sepsis treatment which modulates CXCR-1/2 levels. First, a simplified mechanistic computational model of IL-8 mediated activation of CXCR-1/2 receptors was developed, containing 16 ODEs and 43 parameters. Receptor level dynamics and systemic parameters were coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils which reduce pathogen load, and/or primed neutrophils which cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a two-hour infusion of E coli and followed for a maximum of 28 days. Ensembles of parameters were generated using a Bayesian parallel tempering approach to produce model fits that could recreate experimental outcomes. Stepwise logistic regression identified seven model parameters as key determinants of mortality. Sensitivity analysis showed that parameters controlling the level of killer cell neutrophils affected the overall systemic damage of individuals. To evaluate rescue strategies and provide probabilistic predictions of their impact on mortality, time of onset, duration, and capture efficacy of an extracorporeal device that modulated neutrophil phenotype were explored. Our findings suggest that interventions aiming to modulate phenotypic composition are time sensitive. When introduced between 3-6 hours of infection for a 72 hour duration, the survivor population increased from 31% to 40-80%. Treatment efficacy quickly diminishes if not introduced within 15 hours of infection. Significant harm is possible with treatment durations ranging from 5

Polymorphisms in TAS2R38 and the taste bud trophic factor, gustin gene co-operate in modulating PROP taste phenotype.

Science.gov (United States)

Calò, Carla; Padiglia, Alessandra; Zonza, Andrea; Corrias, Laura; Contu, Paolo; Tepper, Beverly J; Barbarossa, Iole Tomassini

2011-10-24

The PROP taste phenotype varies greatly among individuals, influencing eating behavior and therefore may play a role in body composition. This variation is associated with polymorphisms in the bitter receptor gene TAS2R38 and the taste-bud trophic factor gustin gene. The aim of this study was to examine the relationship between TAS2R38 haplotypes and the gustin gene polymorphism rs2274333 in modulating PROP taste phenotype. PROP phenotype was determined in seventy-six volunteers (29 males, 47 females, age 25±3 y) by scaling methods and threshold measurements. TAS2R38 and gustin gene genotyping was performed using PCR techniques. The lowest responsiveness in PROP nontasters is strongly associated with the AVI nontasting TAS2R38 variant and the highest responsiveness in supertasters is strongly associated to allele A and genotype AA of the gustin gene. These data support the hypothesis that the greater sensitivity of supertasters could be mediated by a greater taste-bud density. Polymorphisms in TAS2R38 and gustin gene, together, accounted for up to 60% of the phenotypic variance in PROP bitterness and to 40% in threshold values. These data, suggest that other unidentified factors may be more relevant for detecting low concentrations of PROP. Moreover, the presence of the PAV variant receptor may be important for detecting high concentrations of PROP, whereas the presence of allele A in gustin polymorphism may be relevant for perceiving low concentrations. These data show how the combination of the TAS2R38 and gustin gene genotypes modulate PROP phenotype, providing an additional tool for the evaluation of human eating behavior and nutritional status. Copyright © 2011 Elsevier Inc. All rights reserved.

Identifying niche-mediated regulatory factors of stem cell phenotypic state: a systems biology approach.

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Ravichandran, Srikanth; Del Sol, Antonio

2017-02-01

Understanding how the cellular niche controls the stem cell phenotype is often hampered due to the complexity of variegated niche composition, its dynamics, and nonlinear stem cell-niche interactions. Here, we propose a systems biology view that considers stem cell-niche interactions as a many-body problem amenable to simplification by the concept of mean field approximation. This enables approximation of the niche effect on stem cells as a constant field that induces sustained activation/inhibition of specific stem cell signaling pathways in all stem cells within heterogeneous populations exhibiting the same phenotype (niche determinants). This view offers a new basis for the development of single cell-based computational approaches for identifying niche determinants, which has potential applications in regenerative medicine and tissue engineering. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

Science.gov (United States)

Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

2017-09-01

Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells

International Nuclear Information System (INIS)

Pinchuk, Lesya M.; Lee, Sang-Ryul; Filipov, Nikolay M.

2007-01-01

Recent data suggest that some of the immunotoxic effects of the herbicide atrazine, a very widely used pesticide, may be due to perturbations in dendritic cell (DC) function. As consequences of atrazine exposure on the phenotypic and functional maturation of DC have not been studied, our objective was, using the murine DC line, JAWSII, to determine whether atrazine will interfere with DC maturation. First, we characterized the maturation of JAWSII cells in vitro by inducing them to mature in the presence of growth factors and selected maturational stimuli in vitro. Next, we exposed the DC cell line to a concentration range of atrazine and examined its effects on phenotypic and functional maturation of DC. Atrazine exposure interfered with the phenotypic and functional maturation of DC at non-cytotoxic concentrations. Among the phenotypic changes caused by atrazine exposure was a dose-dependent removal of surface MHC-I with a significant decrease being observed at 1 μM concentration. In addition, atrazine exposure decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. When, for comparative purposes, we exposed primary thymic DC to atrazine, MHC-I and CD11c expression was also decreased. Phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation as, albeit at higher concentrations, receptor-mediated antigen uptake was increased by atrazine. Thus, our data suggest that atrazine directly targets DC maturation and that toxicants such as atrazine that efficiently remove MHC-I molecules from the DC surface are likely to contribute to immune evasion

Cell-to-module optical loss/gain analysis for various photovoltaic module materials through systematic characterization

Science.gov (United States)

Hsian Saw, Min; Khoo, Yong Sheng; Singh, Jai Prakash; Wang, Yan

2017-08-01

Reducing levelized cost of electricity (LCOE) is important for solar photovoltaics to compete against other energy sources. Thus, the focus should not only be on improving the solar cell efficiency, but also on continuously reducing the losses (or achieving gain) in the cell-to-module process. This can be achieved by choosing the appropriate module material and design. This paper presents a detailed and systematic characterization of various photovoltaic (PV) module materials (encapsulants, tabbing ribbons, and backsheets) and an evaluation of their impact on the output power of silicon wafer-based PV modules. Various characterization tools/techniques, such as UV-vis (reflectance) measurement, external quantum efficiency (EQE) measurement and EQE line-scan are used. Based on the characterization results, we use module materials with the best-evaluated optical performance to build “optimized modules”. Compared to the standard mini-module, an optical gain of more than 5% is achievable for the “optimized module” with selected module materials.

In vitro HgCl2 exposure of immune cells at different stages of maturation: Effects on phenotype and function

International Nuclear Information System (INIS)

Silva, I.A.; Graber, J.; Nyland, J.F.; Silbergeld, E.K.

2005-01-01

This is the first study to investigate the hypothesis that the immunotoxic effects of inorganic mercury may be modulated by inherent differences in the responsiveness of immune cells related to the age of the donor. We exposed cells from lymph nodes, spleen, and thymus, collected from 7- and 10-day-old CD.1 pups, as well as from adult CD.1 mice, in terms of the effects of mercury in vitro on responses to Con-A stimulation with respect to proliferation, cytokine production, and cell phenotype. The effects of mercury on proliferation were age and organ dependent, while effects on cytokine production were only age dependent. Effects of mercury were observed only on splenocyte T-cell subpopulations and only in cells from 10-day-old pups and from adults. Mercury had no effect on IFN-γ and IL-4 production by splenocytes from 7-day-old pups, but significantly decreased release of these cytokines by splenocytes from 10-day-old pups and adults. Hg did not affect IL-4 production by lymph node cells or thymocytes. In lymph node cells Hg affected IFN-γ production only at 7 days. These data indicate that inherent properties of immune cells at different stages of development may influence the response to immunotoxicants

Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

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Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

2017-07-01

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

The phenotype of FancB-mutant mouse embryonic stem cells

OpenAIRE

Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu, Lingchuan; Hasty, Paul

2011-01-01

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslink...

Modulation of photochemical damage in normal and malignant cells by naturally occurring compounds.

Science.gov (United States)

Lee, Yuan-Hao; Kumar, Neeru C; Glickman, Randolph D

2012-01-01

Certain phytochemicals, such as the stilbene, resveratrol (RES, found in red grapes and berries), and the triterpenoid, ursolic acid (UA, found in waxy berries and herbs such as rosemary and oregano), have antioxidant, anti-inflammatory and antiproliferative effects. Two human-derived cell lines, hTERT-RPE with a nonmalignant phenotype derived from retinal pigment epithelium, and ATCC CRL-11147 derived from a malignant skin melanoma, were used as in vitro models of photooxidative stress produced by exposure to the broadband output of a 150 W Hg vapor arc lamp at an irradiance of 19-26 mW cm(-2). In untreated cells, UV-VIS broadband light exposure produced a loss of proliferative ability, an activation of NF-κB and an increase in protein carbonyl adducts at 24 h postexposure. Pretreatment of the cells with RES or UA at 1-2 μmsignificantly reduced the amount of phosphorylated NF-κB at 24 h postexposure. RES pretreatment reduced the burden of light-induced protein carbonyl adducts by up to 25% in exposed cells. UA treatment markedly increased the sensitivity of melanoma cells to UV radiation, while conferring some photoprotection to RPE cells. These observations indicate that phytochemicals modulate the cellular response to photochemical stress by interacting with specific cell-signaling pathways. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Phenotypic Resistance to Antibiotics

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Jose L. Martinez

2013-04-01

Full Text Available The development of antibiotic resistance is usually associated with genetic changes, either to the acquisition of resistance genes, or to mutations in elements relevant for the activity of the antibiotic. However, in some situations resistance can be achieved without any genetic alteration; this is called phenotypic resistance. Non-inherited resistance is associated to specific processes such as growth in biofilms, a stationary growth phase or persistence. These situations might occur during infection but they are not usually considered in classical susceptibility tests at the clinical microbiology laboratories. Recent work has also shown that the susceptibility to antibiotics is highly dependent on the bacterial metabolism and that global metabolic regulators can modulate this phenotype. This modulation includes situations in which bacteria can be more resistant or more susceptible to antibiotics. Understanding these processes will thus help in establishing novel therapeutic approaches based on the actual susceptibility shown by bacteria during infection, which might differ from that determined in the laboratory. In this review, we discuss different examples of phenotypic resistance and the mechanisms that regulate the crosstalk between bacterial metabolism and the susceptibility to antibiotics. Finally, information on strategies currently under development for diminishing the phenotypic resistance to antibiotics of bacterial pathogens is presented.

Melanoma cells revive an embryonic transcriptional network to dictate phenotypic heterogeneity.

Science.gov (United States)

Vandamme, Niels; Berx, Geert

2014-01-01

Compared to the overwhelming amount of literature describing how epithelial-to-mesenchymal transition (EMT)-inducing transcription factors orchestrate cellular plasticity in embryogenesis and epithelial cells, the functions of these factors in non-epithelial contexts, such as melanoma, are less clear. Melanoma is an aggressive tumor arising from melanocytes, endowed with unique features of cellular plasticity. The reversible phenotype-switching between differentiated and invasive phenotypes is increasingly appreciated as a mechanism accounting for heterogeneity in melanoma and is driven by oncogenic signaling and environmental cues. This phenotypic switch is coupled with an intriguing and somewhat counterintuitive signaling switch of EMT-inducing transcription factors. In contrast to carcinomas, different EMT-inducing transcription factors have antagonizing effects in melanoma. Balancing between these different EMT transcription factors is likely the key to successful metastatic spread of melanoma.

Insoluble elastin reduces collagen scaffold stiffness, improves viscoelastic properties, and induces a contractile phenotype in smooth muscle cells.

Science.gov (United States)

Ryan, Alan J; O'Brien, Fergal J

2015-12-01

Biomaterials with the capacity to innately guide cell behaviour while also displaying suitable mechanical properties remain a challenge in tissue engineering. Our approach to this has been to utilise insoluble elastin in combination with collagen as the basis of a biomimetic scaffold for cardiovascular tissue engineering. Elastin was found to markedly alter the mechanical and biological response of these collagen-based scaffolds. Specifically, during extensive mechanical assessment elastin was found to reduce the specific tensile and compressive moduli of the scaffolds in a concentration dependant manner while having minimal effect on scaffold microarchitecture with both scaffold porosity and pore size still within the ideal ranges for tissue engineering applications. However, the viscoelastic properties were significantly improved with elastin addition with a 3.5-fold decrease in induced creep strain, a 6-fold increase in cyclical strain recovery, and with a four-parameter viscoelastic model confirming the ability of elastin to confer resistance to long term deformation/creep. Furthermore, elastin was found to result in the modulation of SMC phenotype towards a contractile state which was determined via reduced proliferation and significantly enhanced expression of early (α-SMA), mid (calponin), and late stage (SM-MHC) contractile proteins. This allows the ability to utilise extracellular matrix proteins alone to modulate SMC phenotype without any exogenous factors added. Taken together, the ability of elastin to alter the mechanical and biological response of collagen scaffolds has led to the development of a biomimetic biomaterial highly suitable for cardiovascular tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

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Tamara Murmann

Full Text Available Small cell lung carcinomas (SCLCs represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Phadke, Manali; Krynetskaia, Natalia [Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Mishra, Anurag [Jayne Haines Center for Pharmacogenomics, Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Krynetskiy, Evgeny, E-mail: ekrynets@temple.edu [Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Jayne Haines Center for Pharmacogenomics, Temple University School of Pharmacy, Philadelphia, PA 19140 (United States)

2011-07-29

Highlights: {yields} We examined the effect of glyceraldehyde 3-phosphate (GAPDH) depletion on proliferation of human carcinoma A549 cells. {yields} GAPDH depletion induces accelerated senescence in tumor cells via AMPK network, in the absence of DNA damage. {yields} Metabolic and genetic rescue experiments indicate that GAPDH has regulatory functions linking energy metabolism and cell cycle. {yields} Induction of senescence in LKB1-deficient lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation. -- Abstract: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-{beta}-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of {alpha} subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based Heterogeneity in Phenotype and Frequency

DEFF Research Database (Denmark)

Simoni, Yannick; Fehlings, Michael; Kloverpris, Henrik N.

2017-01-01

Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors...... to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra-epithelial (ie)ILC1-like cells...... that represent a broader category of NK cells in mucosal and non-mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals...

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

Science.gov (United States)

Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

2018-03-12

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

MicroRNA-93 controls perfusion recovery after hindlimb ischemia by modulating expression of multiple genes in the cell cycle pathway.

Science.gov (United States)

Hazarika, Surovi; Farber, Charles R; Dokun, Ayotunde O; Pitsillides, Achillieas N; Wang, Tao; Lye, R John; Annex, Brian H

2013-04-30

MicroRNAs are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease. Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hindlimb ischemia between inbred mouse strains to identify key microRNAs involved in perfusion recovery from hindlimb ischemia. From comparative microRNA profiling between inbred mouse strains that display profound differences in their extent of perfusion recovery after hindlimb ischemia, we found that the mouse strain with higher levels of microRNA-93 (miR-93) in hindlimb muscle before ischemia and the greater ability to upregulate miR-93 in response to ischemia had better perfusion recovery. In vitro, overexpression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells and enhanced proliferation in both cell types. In addition, miR-93 overexpression enhanced endothelial cell tube formation. In vivo, miR-93 overexpression enhanced capillary density and perfusion recovery from hindlimb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in vitro and in vivo modulation of miR-93 resulted in alterations in the expression of >1 cell cycle pathway gene in 2 different cell types. Our data indicate that miR-93 enhances perfusion recovery from hindlimb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue.

In vitro analysis of the invasive phenotype of SUM 149, an inflammatory breast cancer cell line

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Dharmawardhane Suranganie F

2005-04-01

Full Text Available Abstract Background Inflammatory breast cancer (IBC is the most lethal form of locally invasive breast cancer known. However, very little information is available on the cellular mechanisms responsible for manifestation of the IBC phenotype. To understand the unique phenotype of IBC, we compared the motile and adhesive interactions of an IBC cell line, SUM 149, to the non-IBC cell line SUM 102. Results Our results demonstrate that both IBC and non-IBC cell lines exhibit similar adhesive properties to basal lamina, but SUM 149 showed a marked increase in adhesion to collagen I. In vitro haptotaxis assays demonstrate that SUM 149 was less invasive, while wound healing assays show a less in vitro migratory phenotype for SUM 149 cells relative to SUM 102 cells. We also demonstrate a role for Rho and E-cadherin in the unique invasive phenotype of IBC. Immunoblotting reveals higher E-cadherin and RhoA expression in the IBC cell line but similar RhoC expression. Rhodamine phalloidin staining demonstrates increased formation of actin stress fibers and larger focal adhesions in SUM 149 relative to the SUM 102 cell line. Conclusion The observed unique actin and cellular architecture as well as the invasive and adhesive responses to the extracellular matrix of SUM 149 IBC cells suggest that the preference of IBC cells for connective tissue, possibly a mediator important for the vasculogenic mimicry via tubulogenesis seen in IBC pathological specimens. Overexpression of E-cadherin and RhoA may contribute to passive dissemination of IBC by promoting cell-cell adhesion and actin cytoskeletal structures that maintain tissue integrity. Therefore, we believe that these findings indicate a passive metastatic mechanism by which IBC cells invade the circulatory system as tumor emboli rather than by active migratory mechanisms.

Stability of the phenotypic reversion of x-ray transformed C3H/10T1/2 cells depends on cellular proliferation after subcultivation at low cell density

International Nuclear Information System (INIS)

Brouty-Boye, D.; Gresser, I.; Bandu, M.T.

1982-01-01

Reversion from the transformed to the non-transformed phenotype could be obtained by seeding X-ray transformed C3H/10T1/2 cells at low cell density. Cloned revertant cells of varying degrees of reversion were obtained depending on the time they were isolated after one subculture at low cell density. Most of the revertants isolated 7 and 10 days after seeding at very low cell density eventually returned to the transformed phenotype when passaged serially at high cell density. In contrast, 25-35% of the revertants isolated 17-20 days after seeding at low cell density maintained the non-transformed phenotype despite subsequent serial passages at high cell density. The finding that there was a direct relationship between the time during which transformed cells seeded at low cell density multiplied and the number of stable revertant clones obtained, suggests the possibility that reversion from the transformed to the non-transformed phenotype may be a multistep process. Revertant cells displayed a chromosomal pattern characteristic of the transformed cells rather than that of the parental non-transformed 10T1/2 cells. (author)

Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment.

Directory of Open Access Journals (Sweden)

Al Shaimaa Hasan

Full Text Available Cardiosphere-derived cells (CDCs, one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1 into a regulatory anti-inflammatory phenotype (M2. Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages, but decreased the expression of CD86 (a marker for M1 macrophages 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair.

Phenotypic analysis of perennial airborne allergen-specific CD4+ T cells in atopic and non-atopic individuals.

Science.gov (United States)

Crack, L R; Chan, H W; McPherson, T; Ogg, G S

2011-11-01

Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. © 2011 Blackwell Publishing Ltd.

An AML1-ETO/miR-29b-1 regulatory circuit modulates phenotypic properties of acute myeloid leukemia cells.

Science.gov (United States)

Zaidi, Sayyed K; Perez, Andrew W; White, Elizabeth S; Lian, Jane B; Stein, Janet L; Stein, Gary S

2017-06-20

Acute myeloid leukemia (AML) is characterized by an aggressive clinical course and frequent cytogenetic abnormalities that include specific chromosomal translocations. The 8;21 chromosomal rearrangement disrupts the key hematopoietic RUNX1 transcription factor, and contributes to leukemia through recruitment of co-repressor complexes to RUNX1 target genes, altered subnuclear localization, and deregulation of the myeloid gene regulatory program. However, a role of non-coding microRNAs (miRs) in t(8;21)-mediated leukemogenesis is minimally understood. We present evidence of an interplay between the tumor suppressor miR-29b-1 and the AML1-ETO (also designated RUNX1-RUNX1T1) oncogene that is encoded by the t(8;21). We find that AML1-ETO and corepressor NCoR co-occupy the miR-29a/b-1 locus and downregulate its expression in leukemia cells. Conversely, re-introduction of miR-29b-1 in leukemia cells expressing AML1-ETO causes significant downregulation at the protein level through direct targeting of the 3' untranslated region of the chimeric transcript. Restoration of miR-29b-1 expression in leukemia cells results in decreased cell growth and increased apoptosis. The AML1-ETO-dependent differentiation block and transcriptional program are partially reversed by miR-29b-1. Our findings establish a novel regulatory circuit between the tumor-suppressive miR-29b-1 and the oncogenic AML1-ETO that controls the leukemic phenotype in t(8;21)-carrying acute myeloid leukemia.

High power n-type metal-wrap-through cells and modules using industrial processes

Energy Technology Data Exchange (ETDEWEB)

Guillevin, N.; Heurtault, B.J.B.; Geerligs, L.J.; Van Aken, B.B.; Bennett, I.J.; Jansen, M.J.; Weeber, A.W.; Bultman, J.H. [ECN Solar Energy, P.O. Box 1, NL-1755 ZG Petten (Netherlands); Jianming, Wang; Ziqian, Wang; Jinye, Zhai; Zhiliang, Wan; Shuquan, Tian; Wenchao, Zhao; Zhiyan, Hu; Gaofei, Li; Bo, Yu; Jingfeng, Xiong [Yingli Green Energy Holding Co.,Ltd. 3399 North Chaoyang Avenue, Baoding (China)

2013-10-15

This paper reviews our recent progress in the development of metal wrap through (MWT) cells and modules, produced from n-type Czochralski silicon wafers. The use of n-type silicon as base material allows for high efficiencies: for front emitter-contacted industrial cells, efficiencies above 20% have been reported. N-type MWT (nMWT) cells produced by industrial process technologies allow even higher efficiency due to reduced front metal coverage. Based on the same industrial technology, the efficiency of the bifacial n-MWT cells exceeds the efficiency of the n-type front-and-rear contact and bifacial 'Pasha' technology (n-Pasha) by 0.1-0.2% absolute, with a maximum nMWT efficiency of 20.1% so far. Additionally, full back-contacting of the MWT cells in a module results in reduced cell to module (CTM) fill factor losses. In a direct 60-cell module performance comparison, the n-MWT module, based on integrated backfoil, produced 3% higher power output than the comparable tabbed front emitter-contacted n-Pasha module. Thanks to reduced resistive losses in copper circuitry on the backfoil compared to traditional tabs, the CTM FF loss of the MWT module was reduced by about 2.2%abs. compared to the tabbed front emitter contact module. A full-size module made using MWT cells of 19.6% average efficiency resulted in a power output close to 280W. Latest results of the development of the n-MWT technology at cell and module level are discussed in this paper, including a recent direct comparison run between n-MWT and n-Pasha cells and results of n-MWT cells from 140{mu}m thin mono-crystalline wafers, with only very slight loss (1% of Isc) for the thin cells. Also reverse characteristics and effects of reverse bias for extended time at cell and module level are reported, where we find a higher tolerance of MWT modules than tabbed front contact modules for hotspots.

Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

Science.gov (United States)

Behzad, A R; Morimoto, K; Gosselink, J; Green, J; Hogg, J C; Hayashi, S

2006-12-01

Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.

Phenotypic switching of populations of cells in a stochastic environment

Science.gov (United States)

Hufton, Peter G.; Lin, Yen Ting; Galla, Tobias

2018-02-01

In biology phenotypic switching is a common bet-hedging strategy in the face of uncertain environmental conditions. Existing mathematical models often focus on periodically changing environments to determine the optimal phenotypic response. We focus on the case in which the environment switches randomly between discrete states. Starting from an individual-based model we derive stochastic differential equations to describe the dynamics, and obtain analytical expressions for the mean instantaneous growth rates based on the theory of piecewise-deterministic Markov processes. We show that optimal phenotypic responses are non-trivial for slow and intermediate environmental processes, and systematically compare the cases of periodic and random environments. The best response to random switching is more likely to be heterogeneity than in the case of deterministic periodic environments, net growth rates tend to be higher under stochastic environmental dynamics. The combined system of environment and population of cells can be interpreted as host-pathogen interaction, in which the host tries to choose environmental switching so as to minimise growth of the pathogen, and in which the pathogen employs a phenotypic switching optimised to increase its growth rate. We discuss the existence of Nash-like mutual best-response scenarios for such host-pathogen games.

Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

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Jordan S. Leyton-Mange

2014-02-01

Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: implications for immune cell biobanking.

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Anita Posevitz-Fejfár

Full Text Available Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum, biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9: at a distal location (shipped overnight and in the central laboratory (processed immediately. PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might

Mast Cell Subsets and Their Functional Modulation by the Acanthocheilonema viteae Product ES-62

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Dimity H. Ball

2013-01-01

Full Text Available ES-62, an immunomodulator secreted by filarial nematodes, exhibits therapeutic potential in mouse models of allergic inflammation, at least in part by inducing the desensitisation of FcεRI-mediated mast cell responses. However, in addition to their pathogenic roles in allergic and autoimmune diseases, mast cells are important in fighting infection, wound healing, and resolving inflammation, reflecting that mast cells exhibit a phenotypic and functional plasticity. We have therefore characterised the differential functional responses to antigen (via FcεRI and LPS and their modulation by ES-62 of the mature peritoneal-derived mast cells (PDMC; serosal and those of the connective tissue-like mast cells (CTMC and the mucosal-like mast cells derived from bone marrow progenitors (BMMC as a first step to produce disease tissue-targeted therapeutics based on ES-62 action. All three mast cell populations were rendered hyporesponsive by ES-62 and whilst the mechanisms underlying such desensitisation have not been fully delineated, they reflect a downregulation of calcium and PKCα signalling. ES-62 also downregulated MyD88 and PKCδ in mucosal-type BMMC but not PDMC, the additional signals targeted in mucosal-type BMMC likely reflecting that these cells respond to antigen and LPS by degranulation and cytokine secretion whereas PDMC predominantly respond in a degranulation-based manner.

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

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Cheng Cui

2013-11-01

Full Text Available Planar cell polarity (PCP regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

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Li, Bobby W. S.; Stadhouders, Ralph; de Bruijn, Marjolein J. W.; Lukkes, Melanie; Beerens, Dior M. J. M.; Brem, Maarten D.; KleinJan, Alex; Bergen, Ingrid; Vroman, Heleen; Kool, Mirjam; van IJcken, Wilfred F. J.; Rao, Tata Nageswara; Fehling, Hans Jörg; Hendriks, Rudi W.

2017-01-01

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

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Bobby W. S. Li

2017-12-01

Full Text Available Group 2 innate lymphoid cells (ILC2 are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than

Phenotypic and gene expression changes between low (glucose-responsive) and High (glucose non-responsive) MIN-6 beta cells

DEFF Research Database (Denmark)

O´Driscoll, L.; Gammell, p.; McKierman, E.

2006-01-01

The long-term potential to routinely use replacement beta cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous beta cell studies, by ourselves and other researchers, have indicated...... that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H......, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies...

Solar cell junction temperature measurement of PV module

KAUST Repository

Huang, B.J.

2011-02-01

The present study develops a simple non-destructive method to measure the solar cell junction temperature of PV module. The PV module was put in the environmental chamber with precise temperature control to keep the solar PV module as well as the cell junction in thermal equilibrium with the chamber. The open-circuit voltage of PV module Voc is then measured using a short pulse of solar irradiation provided by a solar simulator. Repeating the measurements at different environment temperature (40-80°C) and solar irradiation S (200-1000W/m2), the correlation between the open-circuit voltage Voc, the junction temperature Tj, and solar irradiation S is derived.The fundamental correlation of the PV module is utilized for on-site monitoring of solar cell junction temperature using the measured Voc and S at a short time instant with open circuit. The junction temperature Tj is then determined using the measured S and Voc through the fundamental correlation. The outdoor test results show that the junction temperature measured using the present method, Tjo, is more accurate. The maximum error using the average surface temperature Tave as the junction temperature is 4.8 °C underestimation; while the maximum error using the present method is 1.3 °C underestimation. © 2010 Elsevier Ltd.

Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome

DEFF Research Database (Denmark)

Pedersen, Martin Bjerregård; Hamilton-Dutoit, Stephen Jacques; Bendix, Knud

2014-01-01

Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome.......Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome....

A probabilistic model for cell population phenotyping using HCS data.

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Edouard Pauwels

Full Text Available High Content Screening (HCS platforms allow screening living cells under a wide range of experimental conditions and give access to a whole panel of cellular responses to a specific treatment. The outcome is a series of cell population images. Within these images, the heterogeneity of cellular response to the same treatment leads to a whole range of observed values for the recorded cellular features. Consequently, it is difficult to compare and interpret experiments. Moreover, the definition of phenotypic classes at a cell population level remains an open question, although this would ease experiments analyses. In the present work, we tackle these two questions. The input of the method is a series of cell population images for which segmentation and cellular phenotype classification has already been performed. We propose a probabilistic model to represent and later compare cell populations. The model is able to fully exploit the HCS-specific information: "dependence structure of population descriptors" and "within-population variability". The experiments we carried out illustrate how our model accounts for this specific information, as well as the fact that the model benefits from considering them. We underline that these features allow richer HCS data analysis than simpler methods based on single cellular feature values averaged over each well. We validate an HCS data analysis method based on control experiments. It accounts for HCS specificities that were not taken into account by previous methods but have a sound biological meaning. Biological validation of previously unknown outputs of the method constitutes a future line of work.

Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

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Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-01-01

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram. PMID:28467792

Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

Science.gov (United States)

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-06-27

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram.

Comparison of GSM Modulated and CW Radiofrequency Radiation on Cells

International Nuclear Information System (INIS)

Pavicic, I.; Marjanovic, A.M.; Trosic, I.

2011-01-01

The aim of our study was to evaluate and compare effect of global system of mobile (GSM) modulation and continuous wave (CW) radiofrequency radiation (RF) on proliferation ability and viability of V79 Chinese hamster lung cells. Previously prepared samples of cells in culture were exposed for 1, 2 and 3 hours both to 915 MHz GSM modulated and to 935 MHz CW RF field in gigahertz transversal electromagnetic mode cell (GTEM-cell). Electric field strength for cells exposed to GSM modulation was set at 10 V/m and for CW exposed cells was 8.2 V/m. Average specific absorption rate (SAR) was calculated to be for GSM 0.23 W/kg and for CW 0.12 W/kg. V79 samples were plated in concentration of 1x10 4 cells/mL. Cell proliferation was determined by cell counts for each hour of exposure during five post-exposure days. Trypan blue exclusion test was used to determine cell viability. In comparison to control cell samples, proliferation of GSM irradiated cells showed significant decrease after 3 hours of exposure on the second and third post-exposure day. CW exposed cell samples showed significant decrease after 3 hours of exposure on the third post-exposure day. Viability of GSM and CW exposed cells did not significantly differ from matched control cell samples. Both applied RF fields have shown similar effect on cell culture growth, and cell viability of V79 cell line. In addition, applied GSM modulated RF radiation demonstrate bigger influence on proliferation of cells. (author)

Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

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Drawnel, Faye M; Boccardo, Stefano; Prummer, Michael; Delobel, Frédéric; Graff, Alexandra; Weber, Michael; Gérard, Régine; Badi, Laura; Kam-Thong, Tony; Bu, Lei; Jiang, Xin; Hoflack, Jean-Christophe; Kiialainen, Anna; Jeworutzki, Elena; Aoyama, Natsuyo; Carlson, Coby; Burcin, Mark; Gromo, Gianni; Boehringer, Markus; Stahlberg, Henning; Hall, Benjamin J; Magnone, Maria Chiara; Kolaja, Kyle; Chien, Kenneth R; Bailly, Jacques; Iacone, Roberto

2014-11-06

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

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Faye M. Drawnel

2014-11-01

Full Text Available Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Cell oxidation-reduction imbalance after modulated radiofrequency radiation.

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Marjanovic, Ana Marija; Pavicic, Ivan; Trosic, Ivancica

2015-01-01

Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800 MHz, strength of 30 V/m on oxidation-reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60 min, specific absorption rate was calculated to be 1.6 W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2',7'-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p < 0.05) increased after 10 min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation-reduction equilibrium within the growing cells.

[Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

Science.gov (United States)

Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

2012-07-01

To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

Real-time tracking of cell cycle progression during CD8+ effector and memory T-cell differentiation.

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Kinjyo, Ichiko; Qin, Jim; Tan, Sioh-Yang; Wellard, Cameron J; Mrass, Paulus; Ritchie, William; Doi, Atsushi; Cavanagh, Lois L; Tomura, Michio; Sakaue-Sawano, Asako; Kanagawa, Osami; Miyawaki, Atsushi; Hodgkin, Philip D; Weninger, Wolfgang

2015-02-24

The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L(hi) central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L(hi) memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.

Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and 'Schizophrenia-Like Behaviors' in Mice.

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Vitucci, Daniela; Di Giorgio, Annabella; Napolitano, Francesco; Pelosi, Barbara; Blasi, Giuseppe; Errico, Francesco; Attrotto, Maria Teresa; Gelao, Barbara; Fazio, Leonardo; Taurisano, Paolo; Di Maio, Anna; Marsili, Valentina; Pasqualetti, Massimo; Bertolino, Alessandro; Usiello, Alessandro

2016-02-01

Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia.

Phenotypic and Functional Alterations in Circulating Memory CD8 T Cells with Time after Primary Infection.

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Matthew D Martin

2015-10-01

Full Text Available Memory CD8 T cells confer increased protection to immune hosts upon secondary viral, bacterial, and parasitic infections. The level of protection provided depends on the numbers, quality (functional ability, and location of memory CD8 T cells present at the time of infection. While primary memory CD8 T cells can be maintained for the life of the host, the full extent of phenotypic and functional changes that occur over time after initial antigen encounter remains poorly characterized. Here we show that critical properties of circulating primary memory CD8 T cells, including location, phenotype, cytokine production, maintenance, secondary proliferation, secondary memory generation potential, and mitochondrial function change with time after infection. Interestingly, phenotypic and functional alterations in the memory population are not due solely to shifts in the ratio of effector (CD62Llo and central memory (CD62Lhi cells, but also occur within defined CD62Lhi memory CD8 T cell subsets. CD62Lhi memory cells retain the ability to efficiently produce cytokines with time after infection. However, while it is was not formally tested whether changes in CD62Lhi memory CD8 T cells over time occur in a cell intrinsic manner or are due to selective death and/or survival, the gene expression profiles of CD62Lhi memory CD8 T cells change, phenotypic heterogeneity decreases, and mitochondrial function and proliferative capacity in either a lymphopenic environment or in response to antigen re-encounter increase with time. Importantly, and in accordance with their enhanced proliferative and metabolic capabilities, protection provided against chronic LCMV clone-13 infection increases over time for both circulating memory CD8 T cell populations and for CD62Lhi memory cells. Taken together, the data in this study reveal that memory CD8 T cells continue to change with time after infection and suggest that the outcome of vaccination strategies designed to elicit

Dual Function of Glucosamine in Gelatin/Hyaluronic Acid Cryogel to Modulate Scaffold Mechanical Properties and to Maintain Chondrogenic Phenotype for Cartilage Tissue Engineering.

Science.gov (United States)

Chen, Chih-Hao; Kuo, Chang-Yi; Wang, Yan-Jie; Chen, Jyh-Ping

2016-11-23

Glucosamine (GlcN) fulfills many of the requirements as an ideal component in scaffolds used in cartilage tissue engineering. The incorporation of GlcN in a gelatin/hyaluronic acid (GH) cryogel scaffold could provide biological cues in maintaining the phenotype of chondrocytes. Nonetheless, substituting gelatin with GlcN may also decrease the crosslinking density and modulate the mechanical properties of the cryogel scaffold, which may be beneficial as physical cues for chondrocytes in the scaffold. Thus, we prepared cryogel scaffolds containing 9% GlcN (GH-GlcN9) and 16% GlcN (GH-GlcN16) by carbodiimide-mediated crosslinking reactions at -16 °C. The crosslinking density and the mechanical properties of the cryogel matrix could be tuned by adjusting the content of GlcN used during cryogel preparation. In general, incorporation of GlcN did not influence scaffold pore size and ultimate compressive strain but increased porosity. The GH-GlcN16 cryogel showed the highest swelling ratio and degradation rate in hyaluronidase and collagenase solutions. On the contrary, the Young's modulus, storage modulus, ultimate compressive stress, energy dissipation level, and rate of stress relaxation decreased by increasing the GlcN content in the cryogel. The release of GlcN from the scaffolds in the culture medium of chondrocytes could be sustained for 21 days for GH-GlcN16 in contrast to only 7 days for GH-GlcN9. In vitro cell culture experiments using rabbit articular chondrocytes revealed that GlcN incorporation affected cell proliferation, morphology, and maintenance of chondrogenic phenotype. Overall, GH-GlcN16 showed the best performance in maintaining chondrogenic phenotype with reduced cell proliferation rate but enhanced glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Quantitative real-time polymerase chain reaction also showed time-dependent up-regulation of cartilage-specific marker genes (COL II, aggrecan and Sox9) for GH-GlcN16. Implantation of

Mechanical modulation of nascent stem cell lineage commitment in tissue engineering scaffolds.

Science.gov (United States)

Song, Min Jae; Dean, David; Knothe Tate, Melissa L

2013-07-01

Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to stem cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to "map the mechanome", defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

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Soares Fernando A

2011-04-01

Full Text Available Abstract Background Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+ and recurrent head and neck squamous cell carcinoma (HNSCC may increase our understanding of the complex biology of this disease. Methods Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative or tumor recurrence (recurrent or non-recurrent tumor after treatment (surgery with neck dissection followed by radiotherapy. Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results The most frequent alterations were the repression of modules in negative lymph node (N0 and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway

C-Si solar cell modules

International Nuclear Information System (INIS)

Tomita, Takashi

2005-01-01

In order to meet the rapidly growing demand for solar power photovoltaic systems which is based on public consciousness of global environmental issues, SHARP has increased the production of solar cells and modules over 10-fold in the last 5 years. Silicon-based technologies are expected to be dominant in the coming decade. In the course of an increase of the annual production scale to 1000 MW, the efficiency of modules will be improved and the thickness of wafers will be decreased and all this will lead to a drastic price reduction of PV systems. (Author)

Formation of photovoltaic modules based on polycrystalline solar cells

OpenAIRE

L. A. Dobrzański; A. Drygała; A. Januszka

2009-01-01

Purpose: The main aim of the paper is formation of photovoltaic modules and analysis of their main electric parameters.Design/methodology/approach: Photovoltaic modules were produced from four polycrystalline silicon solar cells, that were cut and next joined in series. Soft soldering technique and copper-tin strip were used for joining cells.Findings: In order to provide useful power for any application, the individual solar cells must be connected together to give the appropriate current an...

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

Energy Technology Data Exchange (ETDEWEB)

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

Chronic congestive heart failure is associated with a phenotypic shift of intramyocardial endothelial cells

NARCIS (Netherlands)

Marijianowski, M. M.; van Laar, M.; Bras, J.; Becker, A. E.

1995-01-01

There is evidence that patients with chronic congestive heart failure have endothelial cell-related abnormalities of the peripheral circulation and the coronary microvasculature. For that reason, we have studied the phenotypic expression of endothelial cells in hearts of patients with congestive

Trading in your spindles for blebs: the amoeboid tumor cell phenotype in prostate cancer

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Samantha Morley

2014-08-01

Full Text Available Prostate cancer (PCa remains a principal cause of mortality in developed countries. Because no clinical interventions overcome resistance to androgen ablation therapy, management of castration resistance and metastatic disease remains largely untreatable. Metastasis is a multistep process in which tumor cells lose cell-cell contacts, egress from the primary tumor, intravasate, survive shear stress within the vasculature and extravasate into tissues to colonize ectopic sites. Tumor cells reestablish migratory behaviors employed during nonneoplastic processes such as embryonic development, leukocyte trafficking and wound healing. While mesenchymal motility is an established paradigm of dissemination, an alternate, 'amoeboid' phenotype is increasingly appreciated as relevant to human cancer. Here we discuss characteristics and pathways underlying the phenotype, and highlight our findings that the cytoskeletal regulator DIAPH3 governs the mesenchymal-amoeboid transition. We also describe our identification of a new class of tumor-derived microvesicles, large oncosomes, produced by amoeboid cells and with potential clinical utility in prostate and other cancers.

Phenotypic characterization of thymic prelymphoma cells of B10 mice treated with split-dose irradiation

International Nuclear Information System (INIS)

Muto, M.; Kubo, E.; Kamisaku, H.; Sado, T.

1990-01-01

Using an intrathymic injection assay on B10 Thy-1 congenic mice, it was demonstrated that thymic prelymphoma cells first developed within the thymuses from 4 to 8 days after split-dose irradiation and were detected in more than 63% of the test donor thymuses when examined at 21 and 31 days after irradiation. Moreover, some mice (25%) at 2 mo after split-dose irradiation had already developed thymic lymphomas in their thymuses. To characterize these thymic prelymphoma cells, the thymocytes from B10 Thy-1.1 mice 1 mo after irradiation were stained with anti-CD4 and anti-CD8 mAb and were sorted into four subpopulations. These fractionated cells were injected into the recipient thymuses to examine which subpopulation contained thymic prelymphoma cells. The results indicated that thymic prelymphoma cells existed mainly in CD4- CD8- and CD4- CD8+ thymocyte subpopulations and also in CD4+ CD8+ subpopulation. T cell lymphomas derived from CD4- CD8- prelymphoma cells had mainly CD4- CD8- or CD4- CD8+ phenotypes. T cell lymphomas developed from CD4- CD8+ prelymphoma cells mainly expressed CD4- CD8+ or CD4+ CD8+ phenotype. T cell lymphomas originating from CD4+ CD8+ prelymphoma cells were mainly CD4+ CD8+ but some CD4- CD8+ or CD4+ CD8- cells were also present. These thymic prelymphoma cells were further characterized phenotypically in relation to their expression of the marker defined by the mAb against J11d marker and TL-2 (thymus-leukemia) Ag, which is not expressed on normal thymocytes of B10.Thy-1.2 or B10.Thy-1.1 strain, but appears on the thymocytes of lymphomagenic irradiated mice. The results indicated that the prelymphoma cells existed in J11d+, TL-2+ cells

Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

Science.gov (United States)

Li, Pei; Zhang, Ruijie; Wang, Liyuan; Gan, Yibo; Xu, Yuan; Song, Lei; Luo, Lei; Zhao, Chen; Zhang, Chengmin; Ouyang, Bin; Tu, Bing; Zhou, Qiang

2017-04-30

Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells. © 2017 The Author(s).

Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia.

Science.gov (United States)

Bertolino, Alessandro; Fazio, Leonardo; Caforio, Grazia; Blasi, Giuseppe; Rampino, Antonio; Romano, Raffaella; Di Giorgio, Annabella; Taurisano, Paolo; Papp, Audrey; Pinsonneault, Julia; Wang, Danxin; Nardini, Marcello; Popolizio, Teresa; Sadee, Wolfgang

2009-02-01

Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case-control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density.

Phenotypic and functional analysis of CD1a+ dendritic cells from cats chronically infected with feline immunodeficiency virus.

Science.gov (United States)

Zhang, Lin; Reckling, Stacie; Dean, Gregg A

2015-10-01

Numerous studies suggest dendritic cell (DC) dysfunction is central to the dysregulated immune response during HIV infection; however, in vivo studies are lacking. In the present study we used feline immunodeficiency virus (FIV) infection of cats as a model for HIV-1 infection to assess the maturation and function of dendritic cells, in vivo and in vitro. We compared CD1a+ DC migration, surface phenotype, endocytosis, mixed leukocyte reaction (MLR) and regulatory T cell (Treg) phenotype induction by CD1a+ cells isolated from lymph nodes of FIV-infected and control cats. Results showed that resident CD1a+ DC in lymph nodes of chronically FIV-infected cats are phenotypically mature, can stimulate normal primary T cell proliferation, override Treg suppression and do not skew toward Treg induction. In contrast, FIV infection had deleterious effects on antigen presentation and migratory capacity of CD1a+ cells in tissues. Copyright © 2015 Elsevier Ltd. All rights reserved.

Volatility of Mutator Phenotypes at Single Cell Resolution.

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Scott R Kennedy

2015-04-01

Full Text Available Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses.

Science.gov (United States)

Strong, Averey D; Daniels, Richard L

2017-08-02

The tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2. GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student's t-tests and one-way ANOVA. We found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells. We demonstrate the use

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

Science.gov (United States)

Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector

Directory of Open Access Journals (Sweden)

Santhosh Chakkaramakkil Verghese

2016-11-01

Full Text Available Abstract Fanconi anemia (FA is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Comparison of photovoltaic cell temperatures in modules operating with exposed and enclosed back surfaces

Science.gov (United States)

Namkoong, D.; Simon, F. F.

1981-01-01

Four different photovoltaic module designs were tested to determine the cell temperature of each design. The cell temperatures were compared to those obtained on identical design, using the same nominal operating cell temperature (NOCT) concept. The results showed that the NOCT procedure does not apply to the enclosed configurations due to continuous transient conditions. The enclosed modules had higher cell temperatures than the open modules, and insulated modules higher than the uninsulated. The severest performance loss - when translated from cell temperatures - 17.5 % for one enclosed, insulated module as a compared to that module mounted openly.

Clinical phenotypes and the biological parameters of Congolese patients suffering from sickle cell anemia: A first report from Central Africa.

Science.gov (United States)

Mikobi, Tite M; Lukusa Tshilobo, Prosper; Aloni, Michel N; Akilimali, Pierre Z; Mvumbi-Lelo, Georges; Mbuyi-Muamba, Jean Marie

2017-11-01

The influence of phenotype on the clinical course and laboratory features of sickle cell anemia (SCA) is rarely described in sub-Saharan Africa. A cross-sectional study was conducted in Kinshasa. A clinical phenotype score was built up. The following definitions were applied: asymptomatic clinical phenotype (ACP; score≤5), moderate clinical phenotype (MCP; score between 6 and 15), and severe clinical phenotype (SCP; score≥16). ANOVA test were used to compare differences among categorical variables. We have studied 140 patients. The mean body mass index (BMI) value of three groups was lower (Sickle cell patients with ACP have a high mean steady-state hemoglobin concentration compared to those with MCP and SCP (Psickle cell anemia clinical and biological variability in our midst. © 2017 Wiley Periodicals, Inc.

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping

DEFF Research Database (Denmark)

Augustsson, Per; Karlsen, Jonas Tobias; Su, Hao-Wei

2016-01-01

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance...... of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide...... theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic...

Regulatory T cell frequencies and phenotypes following anti-viral vaccination.

Directory of Open Access Journals (Sweden)

A Charlotte M T de Wolf

Full Text Available Regulatory T cells (Treg function in the prevention of excessive inflammation and maintenance of immunological homeostasis. However, these cells may also interfere with resolution of infections or with immune reactions following vaccination. Effects of Treg on vaccine responses are nowadays investigated, but the impact of vaccination on Treg homeostasis is still largely unknown. This may be a relevant safety aspect, since loss of tolerance through reduced Treg may trigger autoimmunity. In exploratory clinical trials, healthy adults were vaccinated with an influenza subunit vaccine plus or minus the adjuvant MF59®, an adjuvanted hepatitis B subunit vaccine or a live attenuated yellow fever vaccine. Frequencies and phenotypes of resting (rTreg and activated (aTreg subpopulations of circulating CD4+ Treg were determined and compared to placebo immunization. Vaccination with influenza vaccines did not result in significant changes in Treg frequencies and phenotypes. Vaccination with the hepatitis B vaccine led to slightly increased frequencies of both rTreg and aTreg subpopulations and a decrease in expression of functionality marker CD39 on aTreg. The live attenuated vaccine resulted in a decrease in rTreg frequency, and an increase in expression of activation marker CD25 on both subpopulations, possibly indicating a conversion from resting to migratory aTreg due to vaccine virus replication. To study the more local effects of vaccination on Treg in lymphoid organs, we immunized mice and analyzed the CD4+ Treg frequency and phenotype in draining lymph nodes and spleen. Vaccination resulted in a transient local decrease in Treg frequency in lymph nodes, followed by a systemic Treg increase in the spleen. Taken together, we showed that vaccination with vaccines with an already established safe profile have only minimal impact on frequencies and characteristics of Treg over time. These findings may serve as a bench-mark of inter-individual variation

Immune modulation through RNA interference-mediated silencing of CD40 in dendritic cells.

Science.gov (United States)

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Samiee, Shahram; Ataee, Zahra; Tabei, Seyyed Ziyaoddin; Moazzeni, Seyed Mohammad

2009-01-01

RNA interference (RNAi) is an exciting mechanism for knocking down any target gene in transcriptional level. It is now clear that small interfering RNA (siRNA), a 19-21nt long dsRNA, can trigger a degradation process (RNAi) that specifically silences the expression of a cognate mRNA. Our findings in this study showed that down regulation of CD40 gene expression in dendritic cells (DCs) by RNAi culminated to immune modulation. Effective delivery of siRNA into DCs would be a reasonable method for the blocking of CD40 gene expression at the cell surface without any effect on other genes and cell cytotoxicity. The effects of siRNA against CD40 mRNA on the function and phenotype of DCs were investigated. The DCs were separated from the mice spleen and then cultured in vitro. By the means of Lipofectamine2000, siRNA was delivered to the cells and the efficacy of transfection was estimated by flow cytometry. By Annexine V and Propidium Iodide staining, we could evaluate the transfected cells viability. Also, the mRNA expression and protein synthesis were assessed by real-time PCR and flow cytometry, respectively. Knocking down the CD40 gene in the DCs caused an increase in IL-4 production, decrease in IL-12 production and allostimulation activity. All together, these effects would stimulate Th2 cytokines production from allogenic T-cells in vitro.

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

International Nuclear Information System (INIS)

Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

2011-01-01

Highlights: ► Isolated ALDH Hi PC3 cells preferentially form primitive holoclone-type colonies. ► Primitive holoclone colonies are predominantly ALDH Lo but contain rare ALDH Hi cells. ► Holoclone-forming cells are not restricted to the ALDH Hi population. ► ALDH phenotypic plasticity occurs in PC3 cells (ALDH Lo to ALDH Hi and vice versa). ► ALDH Hi cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH Lo cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH Hi population, or whether all ALDH Hi cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH Hi cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH Hi cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH Lo population can develop ALDH Hi populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH Hi cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH Hi status enriches for holoclone formation, this activity may be mediated by a minority of ALDH Hi cells.

The Design of a Five-Cell Superconducting RF Module with a PBG Coupler Cell

International Nuclear Information System (INIS)

Arsenyev, Sergey A.; Simakov, Evgenya I.

2012-01-01

We discuss the problem of incorporating a Photonic Band Gap (PBG) cell into a superconducting accelerating module of 5 cells designed for the operational frequency of 2.1 GHz. The reason for using a PBG cell is to provide a good accelerating mode confinement and good Higher Order Mode (HOM) suppression. PBG cell can potentially be used for placing HOM and fundamental mode couplers. However, because of the naturally higher ratio of the peak magnetic field to the accelerating field in the PBG cell, it should be designed to operate at a lower accelerating gradient than the other cells of the module. This ensures that the probability of quench in the PBG cell would be no higher than in other elliptical cells of the structure.

SHOEBOX Modulates Root Meristem Size in Rice through Dose-Dependent Effects of Gibberellins on Cell Elongation and Proliferation.

Science.gov (United States)

Li, Jintao; Zhao, Yu; Chu, Huangwei; Wang, Likai; Fu, Yanru; Liu, Ping; Upadhyaya, Narayana; Chen, Chunli; Mou, Tongmin; Feng, Yuqi; Kumar, Prakash; Xu, Jian

2015-08-01

Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb), a mild gibberellin (GA) deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner.

SHOEBOX Modulates Root Meristem Size in Rice through Dose-Dependent Effects of Gibberellins on Cell Elongation and Proliferation.

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Jintao Li

2015-08-01

Full Text Available Little is known about how the size of meristem cells is regulated and whether it participates in the control of meristem size in plants. Here, we report our findings on shoebox (shb, a mild gibberellin (GA deficient rice mutant that has a short root meristem size. Quantitative analysis of cortical cell length and number indicates that shb has shorter, rather than fewer, cells in the root meristem until around the fifth day after sowing, from which the number of cortical cells is also reduced. These defects can be either corrected by exogenous application of bioactive GA or induced in wild-type roots by a dose-dependent inhibitory effect of paclobutrazol on GA biosynthesis, suggesting that GA deficiency is the primary cause of shb mutant phenotypes. SHB encodes an AP2/ERF transcription factor that directly activates transcription of the GA biosynthesis gene KS1. Thus, root meristem size in rice is modulated by SHB-mediated GA biosynthesis that regulates the elongation and proliferation of meristem cells in a developmental stage-specific manner.

Maternal Adaptive Immune Cells in Decidua Parietalis Display a More Activated and Coinhibitory Phenotype Compared to Decidua Basalis

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Martin Solders

2017-01-01

Full Text Available The maternal part of the placenta, the decidua, consists of maternal immune cells, decidual stromal cells, and extravillous fetal trophoblasts. In a successful pregnancy, these cell compartments interact to provide an intricate balance between fetal tolerance and antimicrobial defense. These processes are still poorly characterized in the two anatomically different decidual tissues, basalis and parietalis. We examined immune cells from decidua basalis and parietalis from term placentas (n=15 with flow cytometry. By using multivariate discriminant analysis, we found a clear separation between the two decidual compartments based on the 81 investigated parameters. Decidua parietalis lymphocytes displayed a more activated phenotype with a higher expression of coinhibitory markers than those isolated from basalis and contained higher frequencies of T regulatory cells. Decidua basalis contained higher proportions of monocytes, B cells, and mucosal-associated invariant T (MAIT cells. The basalis B cells were more immature, and parietalis MAIT cells showed a more activated phenotype. Conventional T cells, NK cells, and MAIT cells from both compartments potently responded with the production of interferon-γ and/or cytotoxic molecules in response to stimulation. To conclude, leukocytes in decidua basalis and parietalis displayed remarkable phenotypic disparities, indicating that the corresponding stromal microenvironments provide different immunoregulatory signals.

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells.

Science.gov (United States)

Guidi, Novella; Sacma, Mehmet; Ständker, Ludger; Soller, Karin; Marka, Gina; Eiwen, Karina; Weiss, Johannes M; Kirchhoff, Frank; Weil, Tanja; Cancelas, Jose A; Florian, Maria Carolina; Geiger, Hartmut

2017-04-03

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging. © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Comparisons of phenotype and immunomodulatory capacity among rhesus bone-marrow-derived mesenchymal stem/stromal cells, multipotent adult progenitor cells, and dermal fibroblasts

Science.gov (United States)

Wang, Qi; Clarkson, Christina; Graham, Melanie; Donahue, Robert; Hering, Bernhard J.; Verfaillie, Catherine M.; Bansal-Pakala, Pratima; O'Brien, Timothy D.

2015-01-01

Background Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4+ and CD8+ lymphocyte populations have not been reported. Methods We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. Results and Conclusions Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable. PMID:24825538

Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

Directory of Open Access Journals (Sweden)

Rust Steven

2013-02-01

Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

Modification of circuit module of dye-sensitized solar cells (DSSC) for solar windows applications

Science.gov (United States)

Hastuti, S. D.; Nurosyid, F.; Supriyanto, A.; Suryana, R.

2016-11-01

This research has been conducted to obtain a modification of circuit producing the best efficiency of solar window modules as an alternative energy for daily usage. Solar window module was constructed by DSSC cells. In the previous research, solar window was created by a single cell of DSSC. Because it had small size, it could not be applied in the manufacture of solar window. Fabrication of solar window required a larger size of DSSC cell. Therefore, in the next research, a module of solar window was fabricated by connecting few cells of DSSC. It was done by using external electrical circuit method which was modified in the formation of series circuit and parallel circuit. Its fabrication used six cells of DSSC with the size of each cell was 1 cm × 9 cm. DSSC cells were sandwich structures constructed by an active layer of TiO2 as the working electrode, electrolyte solution, dye, and carbon layer. Characterization of module was started one by one, from one cell, two cells, three cells, until six cells of a module. It was conducted to recognize the increasing efficiency value as the larger surface area given. The efficiency of solar window module with series circuit was 0.06%, while using parallel circuit was 0.006%. Module with series circuit generated the higher voltage as the larger surface area. Meanwhile, module through parallel circuit tended to produce the constant voltage as the larger surface area. It was caused by the influence of resistance within the cable in each module. Module with circuit parallel used a longer cable than module with series circuit, so that its resistance increased. Therefore, module with parallel circuit generated voltage that tended to be constant and resulted small efficiency compared to the module with series circuit. It could be concluded that series external circuit was the best modification which could produce the higher efficiency.

The Janus-faced role of ezrin in "linking" cells to either normal or metastatic phenotype.

Science.gov (United States)

Brambilla, Daria; Fais, Stefano

2009-11-15

In the majority of eukaryotic cells, the ezrin, radixin and moesin (ERM) proteins are involved in many physiologic functions including regulation of actin cytoskeleton, control of cell shape, adhesion, motility and modulation of signal transduction pathways. In a previous study, we used a dominant negative ezrin-mutant to address ezrin involvement in remodeling of actin cytoskeleton and subsequently we depicted ezrin key role in melanoma cell migration and progression. Herein, we highlight recent advances on ezrin involvement in the metastatic phenomenon, including also some more neglected ezrin-related functions. Novel molecular processes driven by ezrin activation include: phagocytosis, acquisition of resistance to chemotherapeutics and triggering of programmed cell death signals. Recent data support an integrated role of ezrin also in development of tumor malignancy. On one hand, ezrin may be responsible of deranged execution of specific known functions such as adhesion and motility and on the other, it may also participate to unique metastatic determinants, through the establishment of aberrant linkages with tumor-related proteins. For instance, ezrin misslocalization, absence or deranged activity has started to be correlated with tumor progression in many tumors of different species, including humans. Concomitantly, ezrin may act simultaneously as a regulatory or deregulatory chaperon in both normal and tumor cells. It is still to be established whether this Janus-faced feature of ezrin is due to some unknown transforming Zelig-like property or to the fact that a tumor-associated molecule preferentially links to ezrin thus distracting it from its normal connections. However, the contribution of ezrin functional deregulation to the acquisition of the metastatic phenotype appears clear and ezrin or ezrin aberrant associations may represent good candidates for future anti-tumor therapies.

The effect of partial shading on dye-sensitized solar cell module characteristics

International Nuclear Information System (INIS)

Pan, Bin; Weng, Jian; Chen, Shuanghong; Huang, Yang; Dai, Songyuan

2014-01-01

The dye-sensitized solar cell (DSC) is a kind of novel solar cell with prospects for building integrated photovoltaic applications. In some situations, a DSC module may work under partial shading conditions, and subsequently the module temperature and I–V characteristics change. In this work, the effect of partial shading on DSC module characteristics is experimentally studied and the temperature and electric output of the partially shaded DSC module are measured. The variations of module temperature and output performance are analyzed under short circuit conditions and a normal operating mode of charging battery. Furthermore, the stability of the partially shaded DSC module is also evaluated. It is found that the temperature rise of the DSC module caused by partial shading is slower and much smaller than the silicon solar cell, and the characteristics of the single DSC that suffered from short-term shading remain stable. For a DSC module operating in charging mode, the maximum power point and working point change when a shadow appears. (paper)

Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

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Plumas Joel

2009-01-01

Full Text Available Abstract Background Gene modified dendritic cells (DC are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG, the gibbon ape leukaemia virus envelope (GaLV or the feline endogenous virus envelope (RD114. At the same time, we evaluated transgene expression (E-GFP reporter gene under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV under the control of phoshoglycerate kinase (PGK and elongation factor-1 (EF1α promoters (28% to 90% of E-GFP+ cells, respectively in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12 described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

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Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Mao, Lizheng [Jiangsu Asialand Biomed-Technology Co. Ltd., Changzhou, Jiangsu 213164 (China); Deng, Linhong, E-mail: dlh@cczu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Changzhou Key Laboratory of Respiratory Medical Engineering, Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu 213164 (China)

2016-11-15

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

International Nuclear Information System (INIS)

Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong; Mao, Lizheng; Deng, Linhong

2016-01-01

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes a

MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

NARCIS (Netherlands)

Kosten, I.J.; Spiekstra, S.W.; de Gruijl, T.D.; Gibbs, S.

2015-01-01

After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a

Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and ‘Schizophrenia-Like Behaviors' in Mice

Science.gov (United States)

Vitucci, Daniela; Di Giorgio, Annabella; Napolitano, Francesco; Pelosi, Barbara; Blasi, Giuseppe; Errico, Francesco; Attrotto, Maria Teresa; Gelao, Barbara; Fazio, Leonardo; Taurisano, Paolo; Di Maio, Anna; Marsili, Valentina; Pasqualetti, Massimo; Bertolino, Alessandro; Usiello, Alessandro

2016-01-01

Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia. PMID:26228524

PVSIM{copyright}: A simulation program for photovoltaic cells, modules, and arrays

Energy Technology Data Exchange (ETDEWEB)

King, D.L.; Dudley, J.K.; Boyson, W.E.

1996-06-01

An electrical simulation model for photovoltaic cells, modules, and arrays has been developed that will be useful to a wide range of analysts in the photovoltaic industry. The Microsoft{reg_sign} Windows{trademark} based program can be used to analyze individual cells, to analyze the effects of cell mismatch or reverse bias(`hot spot`) heating in modules and to analyze the performance of large arrays of modules including bypass and blocking diodes. User defined statistical variance can be applied to the fundamental parameters used to simulate the cells and diodes. The model is most appropriate for cells that can be accurately modeled using a two-diode equivalent circuit. This paper describes the simulation program and illustrates its versatility with examples.

Effects of Portulaca oleracea L. Polysaccharides on Phenotypic and Functional Maturation of Murine Bone Marrow Derived Dendritic Cells.

Science.gov (United States)

Zhao, Rui; Zhang, Tao; Zhao, Hui; Cai, Yaping

2015-01-01

Portulaca oleracea L. is an annual plant widely distributed from the temperate to the tropical zones. POL-P3b, a polysaccharide fraction purified from Portulaca oleracea L., is able to enhance immunity and inhibit tumor formation. Induction of antitumor immunity by dendritic-tumor fusion cells can be modulated by their activation status. Mature dendritic cells are significantly better than immature dendritic cells at cytotoxic T-lymphocyte induction. In this study, we analyzed the effects of POL-P3b on the maturation and function of murine bone-marrow-derived dendritic cells (DCs) and relevant mechanisms. The phenotypic maturation of DCs was confirmed by flow cytometry. We found that POL-P3b upregulated the expression of CD80, CD86, CD83, and major histocompatibility complex class II molecules on DCs, stimulated production of more interleukin (IL)-12, tumor necrosis factor-α, and less IL-10. Also, DCs pulsed POL-P3b and freeze-thaw antigen increased DCs-driven T cells' proliferation and promoted U14 cells' apoptosis. Furthermore, the expression of TLR-4 was significantly increased on DCs treated by POL-P3b. These results suggested that POL-P3b may induce DCs maturation through TLR-4. Taken together, our results may have important implications for the molecular mechanisms of immunopotentiation of POL-P3b, and provide direct evidence to suggest that POL-P3b should be considered as a potent adjuvant nutrient supplement for DC-based vaccines.

B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

Science.gov (United States)

Hagn, Magdalena; Blackwell, Sue E.; Beyer, Thamara; Ebel, Verena; Fabricius, Dorit; Lindner, Stefanie; Stilgenbauer, Stefan; Simmet, Thomas; Tam, Constantine; Neeson, Paul; Trapani, Joseph A.; Schrezenmeier, Hubert; Weiner, George J.

2014-01-01

CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL. PMID:24497611

Induction of expression of two phenotypic markers of pulmonary type II cells in a cultured cell line

International Nuclear Information System (INIS)

Henderson, R.F.; Waide, J.J.; Scott, G.G.

1994-01-01

The functions of pulmonary type II cells, such as synthesis of pulmonary surfactant and metabolism of inhaled xenobiotics, can be studied in primary isolates of lung cells. However, isolated type II cells, when cultured, quickly lose the phenotypic expressions characteristics of type II cells, including surfactant lipid and protein synthesis and alkaline phosphatase (AP) activity. A cultured cell line that maintained expression of type II cell markers of differentiation would be advantageous for the study of such functions as surfactant synthesis and secretion. Such a cell line would allow generation of a large number of homogeneous cells for study. The purpose of the current study was to induce markers of differentiated type II cells in a cultured cell line to facilitate studies of factors that control surfactant synthesis and secretion

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Institute of Scientific and Technical Information of China (English)

Zhi-Bo Wang; Xiaoqing Zhang; Xue-Jun Li

2013-01-01

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease.Here,we developed a closely representative cell model of SMA by knocking down the disease-determining gene,survival motor neuron (SMN),in human embryonic stem cells (hESCs).Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons.Notably,the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated.Furthermore,these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-A7 (lacking exon 7)knockdown,and were specific to spinal motor neurons.Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes,including specific axonal defects and motor neuron loss.Finally,knockdown of SMNFL led to excessive mitochondrial oxidative stress in human motor neuron progenitors.The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine,a potent antioxidant,which prevented disease-related apoptosis and subsequent motor neuron death.Thus,we report here the successful establishment of an hESC-based SMA model,which exhibits disease gene isoform specificity,cell type specificity,and phenotype reversibility.Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.

CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization

Science.gov (United States)

Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10+ cells. Eight healthy subjects were given a TT booster vacci...

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Science.gov (United States)

Anwar, Adil; Li, Min; Frid, Maria G.; Kumar, Binod; Gerasimovskaya, Evgenia V.; Riddle, Suzette R.; McKeon, B. Alexandre; Thukaram, Roopa; Meyrick, Barbara O.; Fini, Mehdi A.

2012-01-01

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a “constitutively activated” phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the “activated” highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, αVβ3 and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH. PMID:22582113

Minocycline modulates NFκB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

Science.gov (United States)

Guerra, Alberto Daniel; Rose, Warren E; Hematti, Peiman; Kao, W John

2017-07-21

Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds. Modulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague-Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student's t test was used to determine p values for all assays with multiple or two conditions, respectively. Minocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH 2 -terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial

Adult hippocampus derived soluble factors induce a neuronal-like phenotype in mesenchymal stem cells.

Science.gov (United States)

Rivera, Francisco J; Sierralta, Walter D; Minguell, Jose J; Aigner, Ludwig

2006-10-02

Bone marrow-derived mesenchymal stem cells (MSCs) are not restricted in their differentiation fate to cells of the mesenchymal lineage. They acquire a neural phenotype in vitro and in vivo after transplantation in the central nervous system. Here we investigated whether soluble factors derived from different brain regions are sufficient to induce a neuronal phenotype in MSCs. We incubated bone marrow-derived MSCs in conditioned medium (CM) derived from adult hippocampus (HCM), cortex (CoCM) or cerebellum (CeCM) and analyzed the cellular morphology and the expression of neuronal and glial markers. In contrast to muscle derived conditioned medium, which served as control, conditioned medium derived from the different brain regions induced a neuronal morphology and the expression of the neuronal markers GAP-43 and neurofilaments in MSCs. Hippocampus derived conditioned medium had the strongest activity. It was independent of NGF or BDNF; and it was restricted to the neuronal differentiation fate, since no induction of the astroglial marker GFAP was observed. The work indicates that soluble factors present in the brain are sufficient to induce a neuronal phenotype in MSCs.

Endogenous laminin is required for human airway smooth muscle cell maturation

Directory of Open Access Journals (Sweden)

Tran Thai

2006-09-01

Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Doherty, R.E.; Haywood-Small, S.L. [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom); Sisley, K. [Department of Oncology, Academic Unit of Ophthalmology and Orthopties, University of Sheffield, Sheffield S10 2RX (United Kingdom); Cross, N.A., E-mail: n.cross@shu.ac.uk [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

2011-11-04

Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

Solar cell modules with improved backskin and methods for forming same

Science.gov (United States)

Hanoka, Jack I.

1998-04-21

A laminated solar cell module with a backskin layer that reduces the materials and labor required during the manufacturing process. The solar cell module includes a rigid front support layer formed of light transmitting material having first and second surfaces. A transparent encapsulant layer has a first surface disposed adjacent the second surface of the front support layer. A plurality of interconnected solar cells have a first surface disposed adjacent a second surface of the transparent encapsulant layer. The backskin layer is formed of a thermoplastic olefin, which includes first ionomer, a second ionomer, glass fiber, and carbon black. A first surface of the backskin layer is disposed adjacent a second surface of the interconnected solar cells. The transparent encapsulant layer and the backskin layer, in combination, encapsulate the interconnected solar cells. An end portion of the backskin layer can be wrapped around the edge of the module for contacting the first surface of the front support layer to form an edge seal. A laminated solar cell module with a backskin layer that reduces the materials and labor required during the manufacturing process. The solar cell module includes a rigid front support layer formed of light transmitting material having first and second surfaces. A transparent encapsulant layer has a first surface disposed adjacent the second surface of the front support layer. A plurality of interconnected solar cells have a first surface disposed adjacent a second surface of the transparent encapsulant layer. The backskin layer is formed of a thermoplastic olefin, which includes first ionomer, a second ionomer, glass fiber, and carbon black. A first surface of the backskin layer is disposed adjacent a second surface of the interconnected solar cells. The transparent encapsulant layer and the backskin layer, in combination, encapsulate the interconnected solar cells. An end portion of the backskin layer can be wrapped around the edge of the

Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J., E-mail: tokare@niehs.nih.gov

2015-07-01

Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

OpenAIRE

Clémence Roux; Clémence Roux; Clémence Roux; Gaëlle Saviane; Gaëlle Saviane; Jonathan Pini; Jonathan Pini; Nourhène Belaïd; Nourhène Belaïd; Gihen Dhib; Gihen Dhib; Christine Voha; Christine Voha; Christine Voha; Lidia Ibáñez

2018-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for th...

Modulation of phenotype and function of human CD4+CD25+ T regulatory lymphocytes mediated by cAMP elevating agents

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Antonella Riccomi

2016-09-01

Full Text Available We have shown that Cholera Toxin (CT and other cyclic AMP (cAMP elevating agents induce up-regulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP elevating agents on human CD4+CD25+ T cells, which include the T regulatory (Treg cells that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP elevating agents induce up-regulation of CTLA-4 in CD4+CD25- and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3- T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25- T cells did not up-regulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous PBMC. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+ and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP elevating agents induce the up-regulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.

Identification of cancer cytotoxic modulators of PDE3A by predictive chemogenomics

Science.gov (United States)

de Waal, Luc; Lewis, Timothy A.; Rees, Matthew G.; Tsherniak, Aviad; Wu, Xiaoyun; Choi, Peter S.; Gechijian, Lara; Hartigan, Christina; Faloon, Patrick W.; Hickey, Mark J.; Tolliday, Nicola; Carr, Steven A.; Clemons, Paul A.; Munoz, Benito; Wagner, Bridget K.; Shamji, Alykhan F.; Koehler, Angela N.; Schenone, Monica; Burgin, Alex B.; Schreiber, Stuart L.; Greulich, Heidi; Meyerson, Matthew

2015-01-01

High cancer death rates indicate the need for new anti-cancer therapeutic agents. Approaches to discover new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds by phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells while others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggesting a neomorphic activity. Co-expression of SLFN12 with PDE3A correlates with DNMDP sensitivity, while depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery. PMID:26656089

Human lung mast cells modulate the functions of airway smooth muscle cells in asthma.

Science.gov (United States)

Alkhouri, H; Hollins, F; Moir, L M; Brightling, C E; Armour, C L; Hughes, J M

2011-09-01

Activated mast cell densities are increased on the airway smooth muscle in asthma where they may modulate muscle functions and thus contribute to airway inflammation, remodelling and airflow obstruction. To determine the effects of human lung mast cells on the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Freshly isolated human lung mast cells were stimulated with IgE/anti-IgE. Culture supernatants were collected after 2 and 24 h and the mast cells lysed. The supernatants/lysates were added to serum-deprived, subconfluent airway smooth muscle cells for up to 48 h. Released chemokines and extracellular matrix were measured by ELISA, proliferation was quantified by [(3) H]-thymidine incorporation and cell counting, and intracellular signalling by phospho-arrays. Mast cell 2-h supernatants reduced CCL11 and increased CXCL8 and fibronectin production from both asthmatic and nonasthmatic muscle cells. Leupeptin reversed these effects. Mast cell 24-h supernatants and lysates reduced CCL11 release from both muscle cell types but increased CXCL8 release by nonasthmatic cells. The 24-h supernatants also reduced asthmatic, but not nonasthmatic, muscle cell DNA synthesis and asthmatic cell numbers over 5 days through inhibiting extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI3)-kinase pathways. However, prostaglandins, thromboxanes, IL-4 and IL-13 were not involved in reducing the proliferation. Mast cell proteases and newly synthesized products differentially modulated the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Thus, mast cells may modulate their own recruitment and airway smooth muscle functions locally in asthma. © 2011 John Wiley & Sons A/S.

Phenotypic plasticity and epithelial-to-mesenchymal transition in the behaviour and therapeutic response of oral squamous cell carcinoma.

Science.gov (United States)

Vig, Navin; Mackenzie, Ian C; Biddle, Adrian

2015-10-01

It is increasingly recognised that phenotypic plasticity, apparently driven by epigenetic mechanisms, plays a key role in tumour behaviour and markedly influences the important processes of therapeutic survival and metastasis. An important source of plasticity in malignancy is epithelial-to-mesenchymal transition (EMT), a common epigenetically controlled event that results in transition of malignant cells between different phenotypic states that confer motility and enhance survival. In this review, we discuss the importance of phenotypic plasticity and its contribution to cellular heterogeneity in oral squamous cell carcinoma with emphasis on aspects of drug resistance and EMT. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

Science.gov (United States)

Lee, Sangyeop; Chon, Hyangah; Lee, Jiyoung; Ko, Juhui; Chung, Bong Hyun; Lim, Dong Woo; Choo, Jaebum

2014-01-15

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment. © 2013 Elsevier B.V. All rights reserved.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

Science.gov (United States)

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Increased memory phenotypes of CD4+ and CD8+ T cells in ...

African Journals Online (AJOL)

Conclusions: Children with SCA in Tanzania show an absolute increase in all leukocyte types, including lymphocytes, with skewing of both CD4+ and CD8+ T cells towards the memory phenotypes. These findings provide insights on the development of adaptive immunity which may have implications on vaccine ...

Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells

DEFF Research Database (Denmark)

Vaapil, Marica; Helczynska, Karolina; Villadsen, René

2012-01-01

Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less...... is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis....

Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology

Directory of Open Access Journals (Sweden)

Washington R. Cuna

1995-08-01

Full Text Available Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC and cloned. These T cell clones (TCC were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%. On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%, bradycardia with megacolon (75 % and bradycardia (75%. Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

Maternal and fetal mechanisms of B cell regulation during pregnancy: human Chorionic Gonadotropin stimulates B cells to produce IL-10 while alpha-fetoprotein drives them into apoptosis

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Franziska Fettke

2016-12-01

Full Text Available Maternal immune tolerance towards the fetus is an essential requisite for pregnancy. While T cell functions are well documented, little is known about the participation of B cells. We have previously suggested that IL-10 producing B cells are involved in pregnancy tolerance in mice and humans. By employing murine and human systems, we report now that fetal trophoblasts positively regulate the generation of IL-10 producing B cells. We next studied the participation of hormones produced by the placenta as well as the fetal protein alpha-fetoprotein (AFP in B cell modulation. Human Chorionic Gonadotropin (hCG, but not progesterone, estrogen or a combination of both, was able to promote changes in B cell phenotype and boost their IL-10 production, which was abolished after blocking hCG. The hCG-induced B cell phenotype was not associated with augmented galactosylation, sialylation or fucosylation of IgG subclasses in their Fc. In vitro, hCG induced the synthesis of asymmetrically glycosylated antibodies in their Fab region. Interestingly, AFP had dual effects depending on the concentration. At concentrations corresponding to maternal serum levels, it did not modify the phenotype or IL-10 secretion of B cells. At fetal concentrations, however, AFP was able to drive B cells into apoptosis, which may indicate a protective mechanism to avoid maternal B cells to reach the fetus.Our data suggests that the fetus secrete factors that promote a pregnancy-friendly B cell phenotype, unraveling interesting aspects of B cell function and modulation by pregnancy hormones and fetal proteins.

Experimental and theoretical analysis of cell module output performance for a thermophotovoltaic system

International Nuclear Information System (INIS)

Xu, Xiaojie; Ye, Hong; Xu, Yexin; Shen, Mingrong; Zhang, Xiaojing; Wu, Xi

2014-01-01

Highlights: • An accurate theoretical model for thermophotovoltaic system is constructed. • Parallel connected module is superior if radiator temperature is uneven. • Series connected module is superior if cell temperature is uneven. • Short circuit current of series module rises when the shunt resistance decreases. • Fill factor is not always accurate to evaluate the module performance. - Abstract: An experimental thermophotovoltaic (TPV) system with a cylindrical-geometry radiator was established to test the output performances of modules under different conditions. The results demonstrate that the output performance of a cell module decreases when the combustion power increases because of the uneven temperature of the radiator or cells. On this basis, a theoretical model for a TPV system was constructed to compare the performance under different conditions of the series-connected (SC) module and the parallel-connected (PC) module, and was verified by the experimental results. The influences of the temperature gradient of the radiator or the cell module, and the series and shunt resistance of the TPV cell on the module performance were analyzed in detail. The results demonstrate that the PC module can effectively reduce the mismatch loss of output power caused by the uneven radiator temperature. The PC module, for instance, has a maximum output power of 2.54 times higher than that of the SC module when the radiator temperature difference is 500 K. However, the output performance of the module connected in series is superior to the PC module while the cell temperature is non-uniform. The output power of the SC module is 9.93% higher than that of the PC module at the cell temperature difference of 125 K. The short circuit current of the SC module is sensitive to the series and shunt resistance if the radiator temperature distribution is non-uniform. As the shunt resistance falls from ∞ to 0.5 Ω, the current varies from 1.757 A to 4.488 A when the

Operating Cell Temperature Determination in Flat-Plate Photovoltaic Modules

International Nuclear Information System (INIS)

Chenlo, F.

2002-01-01

Two procedures (simplified and complete) to determine me operating cell temperature in photovoltaic modules operating in real conditions assuming isothermal stationary modules are presented in this work. Some examples are included that show me dependence of this temperature on several environmental (sky, ground and ambient temperatures, solar irradiance, wind speed, etc.) and structural (module geometry and size, encapsulating materials, anti reflexive optical coatings, etc.) factors and also on electrical module performance. In a further step temperature profiles for non-isothermal modules are analysed besides transitory effects due to variable irradiance and wind gusts. (Author) 27 refs

Peripheral Tumor Necrosis Factor-Alpha (TNF-α) Modulates Amyloid Pathology by Regulating Blood-Derived Immune Cells and Glial Response in the Brain of AD/TNF Transgenic Mice.

Science.gov (United States)

Paouri, Evi; Tzara, Ourania; Kartalou, Georgia-Ioanna; Zenelak, Sofia; Georgopoulos, Spiros

2017-05-17

Increasing evidence has suggested that systemic inflammation along with local brain inflammation can play a significant role in Alzheimer's disease (AD) pathogenesis. Identifying key molecules that regulate the crosstalk between the immune and the CNS can provide potential therapeutic targets. TNF-α is a proinflammatory cytokine implicated in the pathogenesis of systemic inflammatory and neurodegenerative diseases, such as rheumatoid arthritis (RA) and AD. Recent studies have reported that anti-TNF-α therapy or RA itself can modulate AD pathology, although the underlying mechanism is unclear. To investigate the role of peripheral TNF-α as a mediator of RA in the pathogenesis of AD, we generated double-transgenic 5XFAD/Tg197 AD/TNF mice that develop amyloid deposits and inflammatory arthritis induced by human TNF-α (huTNF-α) expression. We found that 5XFAD/Tg197 mice display decreased amyloid deposition, compromised neuronal integrity, and robust brain inflammation characterized by extensive gliosis and elevated blood-derived immune cell populations, including phagocytic macrophages and microglia. To evaluate the contribution of peripheral huTNF-α in the observed brain phenotype, we treated 5XFAD/Tg197 mice systemically with infliximab, an anti-huTNF-α antibody that does not penetrate the blood-brain barrier and prevents arthritis. Peripheral inhibition of huTNF-α increases amyloid deposition, rescues neuronal impairment, and suppresses gliosis and recruitment of blood-derived immune cells, without affecting brain huTNF-α levels. Our data report, for the first time, a distinctive role for peripheral TNF-α in the modulation of the amyloid phenotype in mice by regulating blood-derived and local brain inflammatory cell populations involved in β-amyloid clearance. SIGNIFICANCE STATEMENT Mounting evidence supports the active involvement of systemic inflammation, in addition to local brain inflammation, in Alzheimer's disease (AD) progression. TNF-α is a

The Natural Killer Cell Cytotoxic Function Is Modulated by HIV-1 Accessory Proteins

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Edward Barker

2011-07-01

Full Text Available Natural killer (NK cells’ major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu’s ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.

Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

Science.gov (United States)

de Groot, Reinoud; Lüthi, Joel; Lindsay, Helen; Holtackers, René; Pelkmans, Lucas

2018-01-23

High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Phenotypic malignant changes and untargeted lipidomic analysis of long-term exposed prostate cancer cells to endocrine disruptors

Energy Technology Data Exchange (ETDEWEB)

Bedia, Carmen, E-mail: carmen.bedia@idaea.csic.es; Dalmau, Núria, E-mail: nuria.dalmau@idaea.csic.es; Jaumot, Joaquim, E-mail: joaquim.jaumot@idaea.csic.es; Tauler, Romà, E-mail: roma.tauler@idaea.csic.es

2015-07-15

Endocrine disruptors (EDs) are a class of environmental toxic molecules able to interfere with the normal hormone metabolism. Numerous studies involve EDs exposure to initiation and development of cancers, including prostate cancer. In this work, three different EDs (aldrin, aroclor 1254 and chlorpyrifos (CPF)) were investigated as potential inducers of a malignant phenotype in DU145 prostate cancer cells after a chronic exposure. Epithelial to mesenchymal transition (EMT) induction, proliferation, migration, colony formation and release of metalloproteinase 2 (MMP-2) were analyzed in 50-day exposed cells to the selected EDs. As a result, aldrin and CPF exposure led to an EMT induction (loss of 16% and 14% of E-cadherin levels, respectively, compared to the unexposed cells). Aroclor and CPF presented an increased migration (134% and 126%, respectively), colony formation (204% and 144%, respectively) and MMP-2 release (137% in both cases) compared to the unexposed cells. An untargeted lipidomic analysis was performed to decipher the lipids involved in the observed transformations. As general results, aldrin exposure showed a global decrease in phospholipids and sphingolipids, and aroclor and CPF showed an increase of certain phospholipids, glycosphingolipids as well as a remarkable increase of some cardiolipin species. Furthermore, the three exposures resulted in an increase of some triglyceride species. In conclusion, some significant changes in lipids were identified and thus we postulate that some lipid compounds and lipid metabolic pathways could be involved in the acquisition of the malignant phenotype in exposed prostate cancer cells to the selected EDs. - Highlights: • Aldrin, aroclor and chlorpyrifos induced an aggressive phenotype in DU145 cells. • An untargeted lipidomic analysis has been performed on chronic exposed cells. • Lipidomic results showed changes in specific lipid species under chronic exposure. • These lipids may have a role in the

Phenotypic malignant changes and untargeted lipidomic analysis of long-term exposed prostate cancer cells to endocrine disruptors

International Nuclear Information System (INIS)

Bedia, Carmen; Dalmau, Núria; Jaumot, Joaquim; Tauler, Romà

2015-01-01

Endocrine disruptors (EDs) are a class of environmental toxic molecules able to interfere with the normal hormone metabolism. Numerous studies involve EDs exposure to initiation and development of cancers, including prostate cancer. In this work, three different EDs (aldrin, aroclor 1254 and chlorpyrifos (CPF)) were investigated as potential inducers of a malignant phenotype in DU145 prostate cancer cells after a chronic exposure. Epithelial to mesenchymal transition (EMT) induction, proliferation, migration, colony formation and release of metalloproteinase 2 (MMP-2) were analyzed in 50-day exposed cells to the selected EDs. As a result, aldrin and CPF exposure led to an EMT induction (loss of 16% and 14% of E-cadherin levels, respectively, compared to the unexposed cells). Aroclor and CPF presented an increased migration (134% and 126%, respectively), colony formation (204% and 144%, respectively) and MMP-2 release (137% in both cases) compared to the unexposed cells. An untargeted lipidomic analysis was performed to decipher the lipids involved in the observed transformations. As general results, aldrin exposure showed a global decrease in phospholipids and sphingolipids, and aroclor and CPF showed an increase of certain phospholipids, glycosphingolipids as well as a remarkable increase of some cardiolipin species. Furthermore, the three exposures resulted in an increase of some triglyceride species. In conclusion, some significant changes in lipids were identified and thus we postulate that some lipid compounds and lipid metabolic pathways could be involved in the acquisition of the malignant phenotype in exposed prostate cancer cells to the selected EDs. - Highlights: • Aldrin, aroclor and chlorpyrifos induced an aggressive phenotype in DU145 cells. • An untargeted lipidomic analysis has been performed on chronic exposed cells. • Lipidomic results showed changes in specific lipid species under chronic exposure. • These lipids may have a role in the

SICKLE CELL DISEASE: REAPPRAISAL OF THE ROLE OF ...